We selected a mutant Paramecium tetraurelia which does not exhibit avoiding reaction in solutions of tetraethylammonium (TEA+), a known membrane K+-channel blocker. Behavioural reaction of the mutant to Na+ solutions was also weak. The rapid successions of avoiding reactions in Ba2+ solutions were observed in both wild type and the TEA-insensitive mutant. Formal genetic analyses showed that this mutant is due to a recessive mutation. This mutation is on a gene completely unlinked to and hypostatic in different degrees to the genes for the membrane defects of ‘pawn A’, ‘pawn B’, ‘ts-pawn C’, ‘fast-2’ and ‘paranoiac A’.

Mutation tam38 of Paramecium tetraurelia is a nuclear recessive mutation with a pleiotropic effect on both trichocyst morphogenesis and nuclear processes. The analysis of the defective nuclear processes (micronuclear and macronuclear divisions, nuclear reorganization at autogamy) shows that these defects result from an abnormal localization of the nuclei. Phenocopies of tam38 abnormalities can be obtained by vinblastine treatment of wild-type cells at late stages of division. Taking into account the similarity between tam38 and a series of other mutations which also prevent trichocyst attachment to the cell surface and disturb nuclear divisions, the following interpretation is proposed: the absence of attached trichocyst induces structural changes in the plasma membrane or in the cortical region which disturb the normal cortical control of the localization of nuclei.

A study of the patterns of phenotypic change in exconjugants using the recessive behavioural mutant pawn (pwA) and its wild-type allele shows that both cytoplasmic and nuclear factors contribute to phenomic lag. Following loss of the wild-type allele from the macronucleus, phenomic lag lasts for 6–11 cell cycles in various sublines of a single clone. Inherited cytoplasmic material is estimated to be responsible for phenomic lag of no more than 5–6 cell cycles. Longer persistence of the parental phenotype is due to continued gene activity in macronuclear fragments carrying the wild-type allele. Genes in fragments remain active and can result in maintenance of the parental phenotype as long as fragments are present (up to 11 cell cycles).

Phenomic lag in the other direction, from pawn to wild type, varies from 0 to 2 cell cycles. The major cytoplasmic factor involved is the amount of wild-type material acquired from the mate during conjugation. Extensive cytoplasmic exchange often occurs during normal conjugation and can lead to change of phenotype as early as the first meiotic division. Phenotypic change due to gene expression in macronuclear anlagen brings about phenotypic change near the end of the first cell cycle in +/+ cells and about a cell cycle later in heterozygotes.

Mating type in Paramecium caudatum, syngen 3 is determined by a pair of alleles with simple dominance; the recessive allele restricts homozygotes to mating type V and the dominant allele permits expression of mating type VI. Clones of mating type V never show natural selfing, but most clones of mating type VI self naturally. A mutant clone of mating type VI which never selfed over a period of more than 3 years was obtained by treatment with N-methyl-N′-nitro-N-nitrosoguanidine. When this mutant clone was crossed to a wild-type stock of mating type V, all F1 clones of mating type VI gave rise to selfers. From selfing of these F1 of mating type VI, clones of F2 were obtained. Nearly 3:1 segregation of selfer to non-selfer clones was observed among the F2 clones of mating type VI. The results were consistent with the interpretation that a dominant modifier gene, Su(+mtV), controls the instability in the expression of mating type VI.

The basis of inheritance of the extra cytoproct (XP) character in Paramecium tetraurelia, stock d4-154, is shown to be nuclear and probably a single dominant gene, Ec, with reduced penetrance in heterozygotes. When the mutant gene is replaced by its wild-type allele, loss of the XP phenotype in some lines of descent occurs before 15 cell generations, but in more than half of the lines this occurs after 15–120 or more cell generations. The possibility is considered that these extremely long and variable ‘lags’ may be due to extranuclear (cortical) inheritance of cortical changes initially produced by gene action.

Caedobacter taeniospiralis (kappa), a bacterial endosymbiont isolated from Paramecium tetraurelia stock 51, contains, in addition to the bacterial chromosome, covalently closed circular DNA molecules as shown by isolation on dye-buoyant-density gradients. The closed circular molecule has a contour length of 13·75 ± 0·04 µm with a buoyant density of 1·698 g/cm3. The buoyant density of the bacterial chromosome is 1·700–1·701 g/cm3. Kappa of the 51 group isolated from stock 298 and stock 6g2, P. tetraurelia, also contain the closed circular DNA. Two forms of kappa coexist in paramecia: brights and nonbrights. Examination by density-gradient centrifugation of the DNA of brights and nonbrights shows the extrachromosomal DNA to be associated mainly with brights. It is suggested that the extrachromosomal DNA might be the determinant for the refractile bodies and the helical phage-like structures found in brights.

A technique has been developed for achieving synchrony of division by selection for immediate pre- and post-fission cells of Paramecium tetraurelia. Cell populations are fed granular tantalum, followed by density-gradient centrifugation. Dividing cells, which do not feed, are separated from feeding interphase cells. The selected cells are division-synchronous for at least two generations when resuspended in fresh culture fluid. This technique provides a rapid means of obtaining large numbers of synchronously dividing paramecia for cell-cycle studies and has general application for selective enrichment of non-feeding sub-populations of cells.

The killing action by mu toxin, which is contained in the cytoplasm of stock 540, Paramecium primaurelia, was demonstrated against the various stocks of paramecia by means of microinjection. Most of the toxin is present in the soluble fraction of the host cytoplasm. The toxin was precipitated by ammonium sulphate at 50–80% saturation, and was almost completely inactivated by incubation at 60 °C for 30 min. Pre-autogamous paramecia were more sensitive than post-autogamous ones to the toxin. Paramecia which bear endosymbionts were generally resistant to the mate-killer toxin.

Oral morphogenesis in stock 51 of Paramecium tetraurelia was investigated using the techniques of Chatton-Lwoff and protargol silver impregnation. During the stomatogenesis accompanying divisional morphogenesis a new oral anlage field and endoral kinety are formed and persist throughout the interfission period in both the proter and the opisthe. This previously overlooked fact is important for understanding the developmental origins and significance of the endoral kinety and the oral anlage field. Previously, the kinetosomes constituting the oral anlage field were thought to be formed just prior to the onset of stomatogenesis, being in some way derived from the kinetosomes of the endoral kinety. The demonstration of a permanent anlage field as an integral component of the oral assemblage suggests that the earliest stages of stomatogenesis might best be viewed as temporally controlled surfacing and/or ciliation of pre-existing kinetosomes rather than their de novo synthesis. The endoral kinety would thus have no contributory role in the formation of the anlage field used in the immediately ensuing stomatogenesis.

Longitudinal microtubules appear in Paramecium tetraurelia when cells are ready to divide (ca. interfission age 0·9). They arise in the longitudinal cortical ridges between kineties and form, in each ridge, an incomplete circlet of 15–18 microtubules, disappearing 10–15 min after separation of the fission products (ca. interfission age 0·03–0·04). Vinblastine, colchicine, colcemid, mercaptoethanol, and cold treatments all result in loss of these microtubules, rounding up of the cells, and some suppression of cell division. The tubules are thought to play a role in cellular elongation, morphogenesis, and separation.

Destruction by laser microbeam of oral and ventral cirral primordia for the future opisthe of 1/2 of a pre-fission doublet results in the permanent loss of ability to form oral and ventral cirral primordia on the irradiated half. This observation provides further evidence for the postulated ‘determinative region’ present on each ventral surface of these organisms. The opisthe of the irradiated doublet retains both original rows of marginal cirri, which are located mid-dorsally with respect to the remaining ventral surface. These two rows of marginal cirri are reproduced faithfully in all future opisthes, but are lost in most proter lines. The pattern of loss in proters involves migration of the extra rows of marginal cirri to the cell's right margin followed by aberrant morphogenesis and subsequent resorption. These results show that the reproduction of marginal cirri is at least partially dependent upon the presence of marginal cirri and their inherent structural characteristics as well as upon their position on the cell surface.

Models of the macronucleus in Paramecium tetraurelia which assume known levels of ploidy and random segregation of subunits smaller than a haploid set at both fission and macronuclear regeneration (MR) are not consistent with the hypothesis that senescence is due to aneuploid imbalance. Either senescence has some other basis or there is a mechanism for regular distribution of subunits at MR. Random segregation models for fission and MR are consistent with most data on survival and heterozygote stability, but if the ploidy level is 860 they fail to account for the data of Nyberg (this volume). Since the ploidy level may be higher than 860, models of random segregation cannot be ruled out for Paramecium. Models of the macronucleus in hypotrichs which assume randomly segregating chromosome fragments are consistent with data on survival and on stability of heterozygous genotypes at fission.

The organization of the genetic material in the macronucleus of ciliates has been the subject of considerable controversy. Two of the four models of macronuclear structure predict assortment of the alternative phenotypes of heterozygotes during vegetative growth. Early studies of the phenotypic behaviour of heterozygotes after macronuclear regeneration (Sonneborn, 1947) had supported a diploid subunit model. The availability of quantitative predictions of the rate of assortment for the haploid and chromosomal models (Preer, this volume) and the existence of two alleles controlling a quantitative trait, copper tolerance, in Paramecium tetraurelia, has provided an opportunity to test these models. The median tolerance limits to copper of unselected sublines were measured as a function of age. There was no increase in the variance among sublines, as the haploid and chromosomal models predict. Quantitative evaluation shows that subdiploid models with a kinetic complexity of 860 or less are not compatible with the results. This experiment was not sensitive enough, however, to exclude subdiploid models if the kinetic complexity is 2000 or greater. Selection on heterozygotes also failed to provide evidence in favour of assortment. All the results are consistent with and support the diploid subunit model of the Paramecium macronucleus.

Genes or sequences of DNA present in multiple copies per cell include entire genomes of mitochondria and chloroplasts, nuclear ribosomal RNA genes, and highly repetitive sequences in heterochromatin. All copies are nearly identical, in spite of mutational pressure and weak selection. A zygote containing mitochondrial or chloroplast genophores of two different genotypes quickly produces progeny pure for one genotype or another (vegetative segregation). Evidence from yeast and Chlamy-domonas suggests that organelle genophores undergo repeated rounds of random mating and recombination. When two molecules carrying different alleles at a locus recombine, gene conversion can result in the cell becoming pure for one allele. Stochastic matching and conversion (SMAC) has been studied by computer simulations which suggest that it will tend to eliminate new mutations in yeast mitochondrial DNA and speed up vegetative segregation. We have verified previous suggestions that gene conversion, occurring during unequal mitotic sister-strand crossing-over, provides an efficient mechanism for maintaining the homogeneity of repeated sequences in eukaryotic chromosomes.

Mating types I and II of Blepharisma japonicum v. intermedium excrete gamones 1 (blepharmone J) and 2 (blepharismone) respectively. The gamone of one type transforms cells of the other type so that they can conjugate with each other. We found that three other species, B. americanum, B. musculus and B. stoltei, have two types of cells homologous to those in B. japonicum; one (type II) excretes a factor which has the same activity as gamone 2 of B. japonicum, the other (type I) responds to this gamone by cell union. Type I cells of these species also excrete a gamone which induces pairs in type II cells of particular strains. Complementarity for mating is observed in some combinations of the two types.

These results indicate that each of the four species has at least one pair of complementary mating types, I and II, with the gamones of the type II's being the same molecule, blepharismone, while gamones of type I's are species- or syngen-specific blepharmone. These generic and specific gamones can be utilized to clarify existing taxonomic and evolutionary questions in the genus Blepharisma.

The macronuclei of Stentor coeruleus were isolated on a discontinuous sucrose gradient and their DNA was purified by conventional methods. The GC content was 32 mole%. The DNA banded as a single peak on analytical ultracentrifugation at 1·691 g/cm3. The molecular weight of the DNA was 5 × 106 to 4 × 107 daltons. Genome size determined by DNA–DNA reassociation kinetics was 6 × 1010 daltons. The macronuclear genome was mostly simple, about 85% being made of non-repetitive sequences.

Activated cysts of Didinium nasutum continue excystment after treatment with actinomycin D or puromycin. Cysts treated at developmental stages 19–20 emerge holociliated and lacking the seizing organ. No effect of the antibiotics at any other stages was found, suggesting that it is at stage 19–20 that the initiation of normal ciliation and of mouth formation occurs. Treatment with other metabolic inhibitors either blocks excystment completely or has no effect on normal development depending upon dosage, suggesting that the action of the antibiotics is specific.

Conjugating pairs of syngen 1 exposed to 0·1 M-CaCl2 during the time of macronuclear development often abort the new macronuclei and retain the old macronucleus and its associated phenotypes. The new micro-nucleus is retained, however, so that macro–micro heterocaryons can be constructed. Because the macronucleus is from a sexually mature strain, the heterocaryon is capable of mating immediately, without passing a period of sexual immaturity. Macronuclear abortion may prove a useful short-cut in genetic analysis.

The two clones produced by the two members of a conjugating pair are normally of the same mating type, about 50% of such synclones being mating type I, 50% type II. This ratio suggests (1) that mating type is determined by a pair of alleles at one locus or by a pair of mating type determining chromosomes, and (2) that one mating type is homozygous, the other heterozygous or hemizygous. Mating type ratios are unaffected by temperature or parental age. Exceptions are of three types: (1) mates that produce one clone of type I, the other of type II, (2) mates which fuse permanently and yield a clone pure for either mating I or mating type II, and (3) conjugation of three mates one of which is regularly non-viable, the two survivors producing either two clones of type I, two clones of type II or one of each type. The bases of these exceptional results remain obscure.