Alpha, an Infectious Macronuclear Symbiont of Paramecium aurelia

SYNOPSIS. A spiral, rod- or crescent-shaped symbiont here designated alpha, is present in the macronucleus of killer stock 562, syngen 2 of Paramecium aurelia. This stock has a cytoplasmic symbiont, kappa, as well as alpha. Lines were obtained which had only alpha, others which had only kappa, and some which had neither. It was possible to purify and separate both kinds of symbiont from homogenates of stock 562 using an ECTEOLA column. The killing action of this stock is due to kappa, not alpha. Observations on the structure of alpha with the electron microscope indicate that alpha, like the cytoplasmic symbionts in this species, is a bacterium. Alpha is never seen in the micronucleus, is rarely found in the cytoplasm, but abounds in the macronucleus. If paramecia are allowed to grow slowly after autogamy, alpha passes from the old macronuclear fragments, infects the new macronucleus, and all animals retain alpha. In exautogamous paramecia growing at maximum fission rate, however, alpha often does not infect the new macronucleus and is lost from many lines when the old macronuclear fragments disappear. In mixed cultures containing alpha-bearing and alphafree paramecia, it has been found that alpha readily invades the macronucleus of paramecia of susceptible stocks. Homogenates of alpha-bearing cultures are also infective. Infection is highly specific, occurring in only 6 of the 44 stocks of P. aurelia in which infection was attempted, and these 6 are all syngen 2. It is suggested that the short rod or crescent form of alpha is the reproductive form, while the elongated spiral form is probably the invasive motile form.     

Studies on the macronucleus of Paramecium aurelia

Following specific incorporation of tritiated thymidine into the DNA of the macronucleus of Paramecium aurelia for varying periods of time the vast majority of silver grains in electron microscope autoradiographs are associated with the area containing the small bodies and matrix. At best only 5% of the silver grains overlying the macronucleus are attributable to the large bodies. This is consistent with the view that the large bodies are the nucleoli of the macronucleus.     

Studies on the surface coat of Paramecium aurelia

Summary The plasma membrane of Paramecium aurelia is covered with a ruthenium red stainable surface coat. Results obtained after digestion with pronase, trypsin and neuraminidase suggest the glycoprotein nature of this structure. Lipid extraction also affects the surface coat forming material. The results are consistent with the model proposed by Ginsburg and Kobata dealing with spatial configuration of the surface coat components.Authors are grateful to Mrs. D. Kucharczyk for very efficient technical assistence, to Mrs. Z. Kaminska for sectioning the material and Mr. A. Renski for help with the electron microscope service.     

Studies on the surface coat of Paramecium aurelia

Summary Correlations between the presence of surface coat and immobilization antigen of Paramecium tetraurelia were studied. Supravital, partial removal of the surface coat resulted in accelerated response of monobacterially and axenically grown cells to the homologous antiserum. Ciliates pretreated with trypsin or pronase (0.5mg/ml for 45 min at 0–4° C) were immobilized approximately twice as fast as untreated control cells. The probable localization of at least part, of the immobilization antigen within the surface coat of P. tetraurelia is discussed.The author thanks Prof. S. Dryl (Department of Cell Biology, M. Nencki Institute of Experimental Biology) and Prof. T.M. Sonneborn (Department of Zoology, Indiana University, Bloomington, USA) for donating antisera and for helpful discussion     

Studies on macronuclear DNA from Paramecium aurelia

Macronuclear DNA was isolated from purified macronuclei of Paramecium aurelia and the size distribution was determined with regard to growth phase and method of extraction. DNA molecules as long as 105 microns and as short as 0.2 microns were observed. It was concluded that the method of extraction affected the observed length of DNA extracted and that macronuclear DNA isolated from cells in balanced growth was less susceptible to nuclease degradation than was DNA isolated from cells in stationary phase. Renaturation studies were performed on macronuclear DNA and a kinetic complexity of 22-times E. coli DNA was determined. This value was similar to those values reported for Tetrahymena and Stylonychia macronuclear DNA. Correcting for GC base content yielded a kinetic complexity for Paramecium macronuclear DNA of 11-times E. coli DNA which corresponded to 3 X 10(10) daltons. There would be about 1400 copies of a unit genome of this complexity within each newly replicated macronucleus. Density gradient analysis indicated that the genes coding for ribosomal RNA had a greater density in CsCl than the bulk DNA. Molecular hybridization studies indicated that the genes coding for 25 S RNA represented 0.14 percent of the total macronuclear DNA. Correcting for GC base content, this corresponded to 30-35 25 S RNA genes per unit genome. These results on Paramecium are discussed in relationship to other ciliate macronuclear DNA.     

Kinetics of Incorporation of DNA Precursors from Ingested Bacteria into Macronuclear DNA of Paramecium aurelia12

SYNOPSIS. The kinetics of transfer of tritium-labeled material from the DNA of ingested bacteria into macronuclear DNA of Paramecium was examined by autoradiography. Bacteria labeled with tritiated thymidine were almost immediately incorporated into food vacuoles, thus becoming available for digestion and a potential source of labeled DNA precursors. Soluble label derived from food vacuoles appeared in low concentrations in the cytoplasm soon after cells were transferred to medium with labeled bacteria; incorporation of labeled precursors into macronuclear DNA began within 5 min. Labeled food vacuoles remained as potential sources of tritiated DNA precursors for a long and variable period after removal of labeled cells to non-labeled medium. The activity of the soluble cytoplasmic DNA precursors decreased parallel to the loss of labeled food vacuoles and no soluble DNA precursors were carried over from one macronuclear DNA synthetic period to the next. Labeling experiments were designed, using this information, which allowed determination of the pattern of macronuclear DNA synthesis and nuclear mass increase during the cell cycle. Macronuclear DNA synthesis began 25–30% of the way thru the cell cycle, continued at a constant rate during the middle half, and decreased in rate during the last quarter. Macronuclear mass increased in an approximately linear fashion, beginning with the onset of DNA synthesis and doubling by the time of karyokinesis.     

Studies on the characterization and structure of the immobilization antigens of Paramecium aurelia

1. Methods for the extraction and purification of the immobilization antigens of Paramecium aurelia have been developed. 2. The immobilization antigen was shown to be a protein of molecular weight 250000. The amino acid content of two allelic antigens and one controlled by a separate genetic locus was determined and many differences were observed. 3. The properties of antigen denatured by reduction and alkylation were examined. The molecular weight fell to 80000 or perhaps less. 4. Peptide maps of allelic types were more similar than those of types controlled by different genetic loci. 5. It is suggested that the antigen consists of two identical half-molecules held together by disulphide bonds. These half-molecules may contain further sub-units.     

Paramecium tredecaurelia of the Paramecium aurelia complex in Israel

The paper concerns the finding of a new habitat (Kiryat Motzkin, north of Haifa, Israel) of Paramecium tredecaurelia from the P. aurelia complex. This is only the forth known locality of the species in the world. Previously, its strains were obtained from widely separated localities: the River Seine, Paris, France; Benenitra, Madagascar, and the Cuernavaca Valley, Taxco, Mexico. The studied strain originating from Israel was identified as P. tredecaurelia on the basis of the strong (90%) conjugation between the complementary mating type of the examined clones with the appropriate standard strain 209 of P. tredecaurelia from Paris, France (restricted to odd mating type). However, the strain from Israel is restricted to the even mating type.     

Mutants Affecting Processing of DNA in Macronuclear Development in Paramecium

In Paramecium tetraurelia, stock 51, the A surface protein is coded by the wild type A51 gene, present in micronuclei in two copies and in macronuclei in about 1500 copies. DNA processing, comprised of DNA cleavage, copy number amplification and telomere addition occurs at autogamy and conjugation when old macronuclei degrade and new macronuclei are formed from micronuclei. In this paper we characterize mutants with macronuclear A gene deletions. These mutants are notable in three respects. First, the mutants do not appear to be simple micronuclear deletions. Although genetic analysis shows that the d12 mutant d12(-1300) is homozygous for the allele A-1300 and the mutant d12(+1) for A+1, analysis by the polymerase chain reaction indicates that the micronuclei in these two mutants contain intact, but presumably altered, micronuclear A genes. They undergo deletion during DNA processing when new macronuclei are formed. Second, the position of the deletions in these alleles has been shown to change. The deficiency present in the d12 allele A-1300 was originally determined to extend from position -1300 (relative to the start of translation of the A gene) to the end of the chromosome. Later, a derivative of this strain, homozygous for the d12 allele A+1 was isolated in which the start site of the deletion was found to have moved from -1300 to +1. Third, a surprising interaction occurs in crosses between a line homozygous for the d12 allele and one homozygous for the wild-type A51 allele. Previous work on the non-Mendelian d48 mutant (which has intact A51 genes in its micronucleus, but has truncated A51 genes in its macronucleus) has shown that intact A51 alleles must be present in the old macronucleus in order for A51 alleles to undergo proper processing. We find that d12 alleles act on A51 alleles in heterozygotes such that intact macronuclear A genes are no longer required for proper processing of A51. Thus, in crosses of 51 x d12 (either +1 or -1300) d12 exconjugants, as well as 51 exconjugants, give rise to clones carrying both intact A51 and truncated d12 alleles. Remarkably the d12 alleles, which are themselves deleted during processing, are capable in the heterozygote of fostering normal processing of the A51 allele.     

Interspecies transfer of mitochondria in Paramecium aurelia.

Summary Erythromycin-resistant mitochondria from species 1, 5 and 7 of P. aurelia were injected into erythromycin-sensitive paramecia of each of the same three species. Mitochondria from species 1 and 5 were successfully transferred to all three species, but species 7 mitochondria failed to develop in species 1 and 5. Minor differences were indicated in the frequency of successful transfers of species 1 mitochondria into species 1 and 5 cells. From studies on the transferability of mitochondria from hybrid cells, containing mitochondria from one species and nuclei from another, it was concluded that mitochondrial compatibility was mainly under control of the nuclear genome, with a possible minor control also by the mitochondrial genome.Dedicated to T.M. Sonneborn on the occasion of his seventieth birthday. This paper is part of a Sonneborn Festchrift most of which will appear in Genetical Research in 1976.     

Species of the Paramecium aurelia complex in Japan.

In Japan, the presence of Paramecium sexaurelia was revealed for the first time, and a new habitat of P. tetraurelia was found there.     

Paramecium sexaurelia of the Paramecium aurelia species complex in Thailand

The clones originating from Thailand, Phuket Island, were identified as Paramecium sexaurelia.