Further Observations on the Chromosomal Filaments of the Ciliate Macronucleus

1966 77 Further Observations on the Chromosomal Filaments of the Ciliate Macronucleus B. R. Seshachar Department of Zoology, University of Delhi Delhi 7, India Received May 7, 1965 Recent reports by the author (1958 , 1960, 1961) indicate that the macronucleus of two species of ciliates , Blepharisma intermedium and Spirostomum ambiguum (Spirotricha) contains greatly elongated Feulgen positive filaments disposed inside the membrane and that by simple methods like stretching the macronucleus , centrifugation and treatment with despiraliz ing agents like KCN and NaCN, it is possible to liberate the filaments from the confines of its membrane. Electron microscope observations of the macronucleus of both species (1964, 1965) have confirmed these observations . Sections show cut-ends of the filaments traversing the macronuclear cavity in all directions. The filaments are composed of microfibrils of about 150 A diameter. The nature of the filaments and the manner of their development are matters of profound interest. Their Feulgen positive nature is indicative of their possessing DNA,-in fact, the macronucleus owes its bright Feulgen positive reaction to them. Their great length is another unique feature. Bundles of them, several mm in length could be obtained from it. The nucleus of no other animal or plant cell has been reported to yield Feulgen positive filaments of such great length by similar treatment. That these filaments are organised structures present inside the macro nucleus and are not artefacts introduced by the methods employed is clear from further studies of the phenomenon. When the cell is cut across and the two parts are gently pulled apart, the macronucleus unwinds itself, producing a thin connection bridging the two parts of the macronucleus (Fig. 1). Individual macronuclei on centrifu gation, often show, along their length, regions which are unwound, alternat ing with intact regions (Fig. 2). This shows that an almost continuous body of filaments is present in the macronucleus some parts of which unwind more easily than the others. It is of special interest in this connection to note that the club-shaped ends of the macronucleus in Blepharisma intermedium are the last to unwind and even when the rest of the nucleus has resolved itself into filaments, their ends remain more or less unaffected. It seems that the filaments are more firmly bound together at the ends than in other regions of the macronucleus. In Spirostomum ambiguum, the macronucleus, 78 B. S. Seshachar Cytologia 31 which consists of a number of connected beads can be stretched to produce a uni form thread of great length. On one occa sion, a thread of more than 6mm was ob tained from a single macro nucleus. It is also of interest that at certain stages of its cycle, the macronucleus does not lend itself to fila ment forma tion. This is especially true of the prefis sion stages. In both Spiro stomunz and Blepharisma, the elongated macronucleus becomes con densed, prior to fission, into a highly com pact body and is later drawn out again dur Figs. 1-3. Blepharisma intermedium. 1, the cell is cut across and the two halves are pulled apart. The thin bridge between the two parts of ing cytokinesis. the macronuclei is clear. Feulgen. 2, a single macronucleus isolated Whatever be from centrifuged material (1800 •~g). The two club-shaped ends are close together at arrow. The macronucleus has unwound itself in certain the significance regions. At other regions, the macronucleus is intact. Feulgen. 3, of this con part of macronucleus from centrifuged material (1800•~g) showing the fi lament issuing from it. Its disposition inside the membrane is also clear. Feulgen. 1966 Further Observations on the Chromosomal Filaments of the Ciliate Macronucleus 79 densation, it is of interest that the macronucleus in its condensed condition cannot be induced, by centrifugation or mechanical manipulation , to produce filaments. It is very striking to see in centrifuged material , amidst a mass of filaments produced by vegetative nuclei , highly condensed and unchanged prefission nuclei. A study under the electron microscope, of the macro nucleus in different stages of its division cycle should be highly interesting . That the filaments are present inside the macronucleus and that they issue from it under conditions described in this paper is further illustrated in Fig. 3 which is a part of the macronucleus of B . inztermedium. The filament can be traced back into the macronucleus and its disposition inside is clearly discernible. The above observations confirm the view that the macronucleus in these two species contains filaments of great length which are disposed inside the membrane in all directions. Electron microscope observations in both species have shown that outside these filaments, the macronucleus has nothing but the nucleoli (Seshachar 1964, 1965). These filaments represent the chromo somes of the macronucleus. Summary Further proof is provided for the view that the chromosomes in the macronucleus of Blepharisma and Spirostomum are in the nature of organized Feulgen positive filaments disposed within its membrane. Under appropriate conditions, the filaments unwind themselves from the macronucleus to give rise to greatly elongate structures. Literature cited Seshachar, B. R. 1958. The macronucleus of Spirostomum. Nature, London 182: 1614-15. - 1960. Effect of centrifugation on the macronucleus of Spirostomum and Blepharisma. Nature, London 186: 333-334.- 1961. Experiments with the Ciliate macronucleus. Progress in Protozoology. Proceed ings of the 1st International Conference on Protozoology, Prague: 275-279. - 1964. Observations on the fine structure of the nuclear apparatus of Blepharisma intermedium Bhandary (Ciliata: Spirotricha). J. Protozool. 11: 402-409. - 1965. The fine structure of the nuclear apparatus and the chromosomes of Spirostomum ambiguum Ehrbg. Acta Protozoologica 3: 337-343..

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