Mating‐type dependent cell pairing in Oxytricha hymenostoma. IV. Macronuclear DNA content

Abstract

The cytophotometric study of vegetative cells of Oxytrichu hymenostoma showed that the two complementary strains 295 and 301 differ from each other in their macronuclear DNA content: 902 arbitrary units (U) for 295 and 1365 U for 301.During paraconjugation, a phenomenon involving the total fusion of two complementary partners into a unique, new cellular entity, the macronuclear DNA contentlpair does not change strikingly: for the first 3 hr, in fact, the macronuclear DN.A content per pair ranges around 2250 U, such a value being very close to that of a 295 cell (902 U) plus that of a 301 cell (1365 U). Only at stages 6 and 7, an increase of the macronuclear DNA content occurs (+9%), which is likely to be due to ano:malous replication bands. Finally, at stages 8 and 9, the macronuclear DNA contentlpair progressively decreases, dropping to values as small as 2025 U, in spite of the apparently normal replication bands, passing through the macronuclear, soon before the first binary fission of the paraconjugant cell.Previous cytologic and ultrastructural analyses of the sexual reproduction of Oxytricha hymenostoma (Esposito et al., '78; Banchetti et al., '80a) showed that cells belonging to the complementary types 295 and 301 undergo a peculiar, total cell fusion, defined "paraconjugation" (Banchetti et al., '80b). The main traits of this process are (a) the lack of micronuclear meiosis and (b) the fusion of the macronuclear pieces of the two partners, with the formation of a unique cellular entity with an apparently normal cortical, cytoplasmic, and nuclear morphology. By genetic analysis (Banchetti et al., 'Sob) it was concluded that: (1) the observed mating-type assortment in the progeny indicates a macronuclear segregation and (2) the over-representation of 301 among the progeny cannot be ascribed to an imperfect mixing of macronuclear material, according to the electron microscopy examination (Banchetti et al., '80a).Does any preparaconjugative regulation of the DNA content take place? Does any postparaconjugative regulation of the DNA content take place? In an attempt to answer these questions, and on the basis of the results already acquired, a cytophotometric analysis of paraconjugation in 0. hymenostoma was conducted t o study the trend of the macronuclear DNA content during the various stages of the process. MATERIALS AND METHODSThe two strains RN3 and RBI, belonging to the complementary mating types of 295 and 301 of 0. hymenostoma, were used for this round of experiments. The cultures were grown according to the improved culturing techniques, already standardized for Oxytn'cha bifun'a (Ricci et al., '80a), and kept at a temperature of about 23 k 1°C. After a mild starvation, about 10-12 hr, cells of both mating types were mixed together and the pairs were isolated as soon as they formed. Every 30 min, from 0 to 9 hr, pairs of equal age were fixed by Sanfelice fixative (cromic acid 1% aqueous solution, 16 parts; Formalin, 8 parts; glacial acetic acid, 1 part), attached to the slides with 95% (vlv)...