Cortical Characteristics of Strains of Syngens 10, 11 and 12 of Tetrahymena pyriformis*

SYNOPSIS. The patterns of correlation among cortical structures have been examined in strains of 3 additional breeding groups (syngens) of Tetrahymena pyriformis. As in previous studies, many cytogeometric properties were found to vary within a syngen. Surprisingly, one strain of syngen 12 differed from 3 others in the one property previously found to be constant—the pattern of variation of the position of the contractile vacuole pores. This strain may, however, be a sexually cross-reacting representative of still another syngen. This interpretation would preserve the diagnostic value of CVP position, and permit an ordering of syngens in terms of the distance of the CVP's from meridian #1: 1, 3, 7, 6, 8, 11, 12, 9, 2, 5, 4, 10.     

Corticotypic Technics in Tetrahymena Taxonomy*

SYNOPSIS. Strains of Tetrahymena pyriformis have a cryptic polymorphism reflected in the production of individuals with different numbers of ciliary rows (meridians). Within a strain certain cortical features—particularly the numbers and positions of contractile vacuole pores, the numbers of postoral meridians, and the stability of the cell type-vary in a systematic way with meridianal multiplicity. A preliminary survey of strains representing 10 genetically isolated groups reveals several patterns of correlation of cortical elements. All the groups examined appear to be distinctive with the possible exception of the strains from syngens 6 and 8, which are known to be genetically compatible under certain conditions. These studies encourage a belief that the heterogeneous assembly of genetic species now sheltered under the taxonomic umbrella of T. pyriformis can soon be given independent morphologic identification and appropriate specific designations.     

The Stability of Cortical Phenotypes in Continuously Growing Cultures of Tetrahymena pyriformis*

Cortical features were analyzed in successive samples of continuously growing stock cultures of amicronucleate strains GL-C and GL-I, and in micronucleate strain WH-6 (syngen 1, mating type I). Thirteen successive samples of strain GL-C, representing a time span of 111 months, 5 samples of WH-6 (43 months) and 2 samples of GL-I (1 month) were examined. The observed range of commonly expressed ciliary row numbers (corticotypes) was 16–20 rows in strain GL-C, 15–20 in strain GL-I, and 16–20 in strain WH-6. These ranges remained constant through time within each strain. The individual samples each included all or a large part of the total range observed in the strain, but the relative abundances of different corticotypes within this range shifted through time. The shifts appeared random, with no discernible trends. Mean contractile vacuole pore (CVP) position and number of CVP meridians were assayed in the 2 “GL” strains. Mean CVP position was an apparently stable character, with only slight fluctuations through time, while the distribution of number of CVP meridians was somewhat less constant. The CVP parameters of strains GL-C and GL-I were considerably different, and both of these strains were very different from the GL strain which had been studied by Nanney. In fact, these 3 “GL” strains have, among them, virtually the entire gamut of known CVP characteristics. The possible significance of these wide differences among strains presumed to be closely related is considered in the Discussion.     

Patterns of Cortical Stability in Tetrahymena*

SYNOPSIS. A simplified technic is presented for determining clonal stability patterns for numbers of ciliary rows in Tetrahymena. The technic involves plotting the variance of meridian number against the mean number of meridians. By this procedure strains of different syngens, difficult to discriminate on other grounds, may sometimes be shown to be distinctive.     

Enzyme Variation in Paramecium caudatum

Stocks of four “syngens” (syngens 1, 3, 12, 13) of Paramecium caudatum could not be distinguished on the basis of isozymic variations of six enzymes. Using the most common enzyme form as the standard for the syngen, we found a higher coefficient of identity between syngens (>90%) than within syngens (73%). These observations, combined with evidence for fertile matings among the groups, suggest that the groups do not warrant the status of syngens. True syngenic variation in P. caudatum is, however, clearly established on the basis of isozymic and breeding studies of wild stocks collected from various places. Some stocks from England have a close affinity with those from Japan, but stocks from Scotland and California must be placed in separate syngenic sets. Thus, P. caudatum is indeed a species complex, within which evolutionarily distinct groups (species = syngens) are identifiable. The definition of syngens on the basis of initial agglutination response is, however, unjustified.     

Intersyngenic variations in the esterases of axenic stocks of Paramecium aurelia

The esterase isozymes were surveyed in axenic stocks of syngens 1, 2, 4, 5, 6, and 8 of Paramecium aurelia by starch gel electrophoresis. In paramecia there appear to be four types of esterases which are clearer in axenic than in bacterized stocks. Each type differs in its substrate specificity and/or its response to the inhibitor eserine sulfate. Minor variations in type D esterases sometimes occur in different extracts of the same stock and may result from changes in the temperature of growth of the cells or growth cycle differences. Differences in the mobility of the A, B, or C (cathodal) types of esterases may occur in different syngens. They also occur for the A and B types among stocks within a syngen, but the frequency is low, except in the case of syngen 2. Since each of the types of esterases varies independently, at least four and possibly more genes appear to specify the esterases in the species complex. Some pairs of syngens vary in their electrophoretic positions for all types of esterases. Other pairs have identical zymograms. This observation suggests that some syngens may differ from each other by as many as four esterase genes, while others may not differ at all. The difference between P. aurelia and Tetrahymena pyriformis in the degree of intrasyngenic variation observed for enzymes is discussed in relation to other types of characters, the organization of the genetic material in the macronucleus, the presence of symbionts, and their breeding systems. It is suggested that enzyme variation is achieved by the action of different selective forces in these two groups of ciliated protozoa.Supported by research grants from the National Institute of General Medical Sciences (GM-15879), U.S. Public Health Service, and from the British Medical Research Council.     

Enzyme variation between syngens in Paramecium aurelia

Five enzymes (succinic dehydrogenase isocitrate dehydrogenase, glutamate dehydrogenase, -hydroxybutyrate dehydrogenase, and fumarase) in the ciliate Paramecium aurelia have been examined by starch gel electrophoresis. Relatively few variants were found among stocks belonging to a given syngen and only in two out of the five enzymes studied. Comparison of stocks belonging to different syngens, however, revealed many enzyme differences, which did not resemble intrasyngen variants. By studying the electrophoretic patterns of the enzymes of the 14 described syngens of Paramecium aurelia, it was found that only two syngens (Nos. 1 and 5) were indistinguishable. It is concluded that the use of starch gel electrophoresis of suitable enzymes provides a method of assigning a stock of paramecia to its syngen. Such a method may, in some cases, be less laborious than the standard one of making test matings and would, of course, be available for organisms which show no mating reaction.This work was carried out during the tenure of an M.R.C. Postgraduate Research Scholarship, and forms part of the material for a Ph.D. thesis at the University of Edinburgh.     

SEXUAL AND GENETIC ISOLATION IN THE COSMOPOLITAN ALGAL SPECIES PANDORINA MORUM

In an effort to analyze the nature of a taxonomic species in the microalgae, the major biological characters of various isolates of Pandorina morum have been studied. Seventy heterothallic clones from 31 collecting sites on three continents were found to represent 20 reproductively isolated groups, based on their ability to form zygotes when paired in all possible combinations. Successful germination of the zygotes and demonstrated fertility of their products suggest that each reproductively isolated group represents a syngen—a group of organisms sharing a common gene pool. A further indication of the close relationship of members of the same syngen is that its representatives all share a common chromosome number, time of division, and zygote distribution pattern, characters which vary between syngens. The known range of 18 of the syngens is less than 500 km. The other two syngens may have a world-wide distribution; they both have a haploid chromosome number of 5. Crosses between geographically isolated clones in these two syngens and one other give evidence of both pre- and postzygotic partial isolation within a syngen. Analysis of collections from two revisited sites demonstrated that representatives of a syngen can be recovered after 19 years have elapsed, and that a single pond may contain as many as four syngens. The results obtained for P. morum are compared with the information available on other microalgae with a similar breeding system, and the possible causes and consequences of the species structure are discussed.     

Cortical Patterns in Two Syngens of Glaucoma*

SYNOPSIS. Strains of 2 syngens of Glaucoma (16) were found to have different cortical characteristics. All clones of a particular strain had approximately the same range of corticotypes, approximately the same means and high and very similar variances. The chief differences between the syngens was the corticotypic range. The patterns of variation of cortical elements of syngen 2 appeared to be primarily extensions of the patterns of syngen 1. The range of meridian numbers of different species of Glaucoma overlapped with each other and could not be distinguished by this criterion alone.     

Comparison of the Esterases and Acid Phosphatases in Paramecium multimicronucleatum,Syngens 1–5, P. jenningsi,P. caudatum,and the P. aurelia Complex1

ABSTRACT. Forty-eight stocks in Paramecium jenningsi, syngens 1–5 of P. multimicronucleatum, P. caudatum, P. primaurelia, P. biaurelia, and P. tetraurelia were grown axenically and tested for their esterases and acid phosphatases using starch gel electrophoresis. The five esterases and the acid phosphatases previously characterized in species of the P. aurelia complex were also found in P. jenningsi, and three to four of the esterases and the acid phosphatases were found in the P. multimicronucleatum species complex and in P. caudatum. Additional subtypes were observed for each of the enzyme phenotypes in these new (though here unnamed) species of Paramecium. Two of the new acid phosphatase subtypes, which depart radically in mobility and in pattern, were found in syngen 3 of P. multimicronucleatum and in P. caudatum. Except for syngens 1 and 5 in P. multimicronucleatum, the degree of similarity between syngens 1, 5 and 2, 3, and 4 appears to be very low—perhaps even lower than that seen for species in the aurelia complex. More realistically, the syngens of P. multimicronucleatum should be considered as separate species although they are not here given separate taxonomic names. Limited sharing of subtypes occurred between species in different species complexes. This observation suggests that the molecular distances between species complexes may be even greater than between species within a complex.     

Intersyngenic variations in the esterases and acid phosphatases of Tetrahymena pyriformis

The esterase and acid phosphatase isozymes were surveyed in strains of syngens 2–12 under conditions found to be optimal for syngen 1. Both intersyngenic and intrasyngenic variations were found. Comparisons of the esterases suggest that homologous enzymes are present in certain syngens and that some ordering of the variations with respect to syngen differences is possible. The acid phosphatases are highly polymorphic in different strains even within a syngen, and the variations cannot be ordered with respect to syngen differences. These results are discussed in terms of other types of studies directed at assessing syngen relationships and in terms of the sources of variation. It was concluded that only characters less vulnerable to intraclonal variation will be capable of revealing syngen relationships.Supported by a Research Grant, GM-15879, from the National Institute of General Medical Sciences, U.S. Public Health Service.     

Identification of Paramecium bursaria syngens through molecular markers--comparative analysis of three loci in the nuclear and mitochondrial DNA

This is the first attempt to resolve the phylogenetic relationship between different syngens of Paramecium bursaria and to investigate at a molecular level the intraspecific differentiation of strains originating from very distant geographical locations. Herein we introduce a new collection of five P. bursaria syngens maintained at St Petersburg State University, as the international collection of syngens was lost in the 1960s. To analyze the degree of speciation within Paramecium bursaria, we examined 26 strains belonging to five different syngens from distant and geographically isolated localities using rDNA (ITS1-5.8S-ITS2-5'LSU) fragments, mitochondrial cytochrome c oxidase subunit I (COI), and H4 gene fragments. It was shown that P. bursaria strains of the same syngens cluster together in all three inferred molecular phylogenies. The genetic diversity among the studied P. bursaria strains based on rDNA sequences was rather low. The COI divergence of Paramecium bursaria was also definitely lower than that observed in the Paramecium aurelia complex. The nucleotide sequences of the H4 gene analyzed in the present study indicate the extent of genetic differences between the syngens of Paramecium bursaria. Our study demonstrates the diagnostic value of molecular markers, which are important tools in the identification of Paramecium bursaria syngens.     

Genetic control of lactate dehydrogenase isozymes in Paramecium caudatum

The LDH isozymes were surveyed in bacterized cultures of syngens 1, 3, 12, and 13 of Paramecium caudatum by polyacrylamide gel electrophoresis. All the examined stocks of different syngens except one stock in syngen 3 had a single common LDH isozyme, and intra- and intersyngenic variation was not observed except for the one stock in syngen 3. Breeding data using the exceptional stock indicated that the LDH isozymes of P. caudatum are controlled by two codominant alleles at a single locus whose products aggregate randomly, forming a dimer.     

The Purification of DNA from the Genomes of Paramecium aurelia and Tetrahymena pyriformis1

SYNOPSIS. Methods are described for the recovery of highly purified DNA from Paramecium aurelia and Tetrahymena pyriformis in high yields. Our DNA is only slightly contaminated with RNA and carbohydrate, and little or no protein can be detected. We could not reduce the RNA (orcinol-positive material) by further treatment (sephadex or hydroxyapatite chromatography and preparative CsCl gradients). At the extreme our DNA is contaminated with 15–20% RNA but the real value is most likely considerably lower than this. The DNA we have prepared from Paramecium and Tetrahymena shows all the properties of double-stranded, high molecular weight DNA when characterized by temperature melting, CsCl density gradient centrifugation and hydroxyapatite and sephadex chromatography. When denatured, it absorbs to nitrocellulose filters. The 2 major results of importance from our work reported here are: (1) There is similarity in base composition of DNA from different syngens of Paramecium (28% G+C for syngens 1, 2, 4, 5, and 9 and 29–30% G+C for syngen 8) while there is variation between the syngens of Tetrahymena (24–31% G+C for syngens 1, 4, 7, 10, 11, and 12); (2) the density of any Paramecium DNA varies depending upon whether the cells are grown in the presence of bacteria or in axenic medium. Our results are compatible with observations previously reported for Tetrahymena but contradict those made for Paramecium. The earlier reports of differences in base composition between syngens of Paramecium are probably due in large part to the use of stocks grown on bacteria.     

MOLECULAR DELINEATION OF SPECIES AND SYNGENS IN VOLVOCACEAN GREEN ALGAE (CHLOROPHYTA)1

Two species of the colonial green flagellate family Volvocaceae are worldwide in distribution yet exhibit contrasting species structure. Geographically disparate isolates of Gonium pectorale Mueller can interbreed while isolates of Pandorina morum Bory behave quite differently. More than 20 sexually isolated subpopulations occur within this species; these have been termed “syngens” (sensu Sonneborn). Because prezygotic barriers to mating cause intersyngen pairings to fail, breeding analyses cannot be used to estimate genetic relatedness among the syngens of P. morum. DNA comparisons provide an alternative method of assessing genetic relatedness. We compared the nucleotide sequence of the internal transcribed spacer (ITS) region of the nuclear ribosomal repeat among clones of P. morum and of G. pectorale. Members of syngens of P. morum with distribution restricted to one small geographical area show great similarity. Likewise, members of any syngen of worldwide distribution show near uniformity, even those from different continents. However, the ITS sequence of each syngen differs from that of other syngens. In contrast, G. pectorale, which has an ITS region that is remarkably uniform throughout the world, appears to consist of a single syngen within North America and Europe by mating tests. The molecular data are in complete conformity with previous syngen assignment. Because the latter is based on mating affinity, with two complementary mating types per syngen, the evolution of new mating type pairs appears to be the basis of microevolution in these algae. We infer that either P. morum is a more ancient species than G. pectorale or that P. morum has a less stable genome. In either case, the biogeographic distribution of certain syngens may reflect climatological changes of the past.     

Intersyngenic variations in the esterases of bacterized Paramecium aurelia

The esterase isozymes were surveyed in bacterized stocks representative of all 14 syngens of Paramecium aurelia by starch gel electrophoresis. The properties of substrate specificity and independent variation of particular isozymes permit the ordering of the differences observed among stocks. Differences can arise from several sources: bacterial variation, intrasyngenic variation, and intersyngenic variation. Bacterial esterases tend to be found in certain zonal areas (see Rowe et al., 1971) and produce minor stock differences, which are erratic in their distribution. Unlike the situation found in Tetrahymena pyriformis, major intrasyngenic variations are rare in P. aurelia except in syngen 2. This lack of intrasyngenic variation is significant in view of the wide differences in geographic origin and micronuclear chromosome numbers among stocks within a syngen. It suggests that certain esterase genotypes must be under stringent selection within a syngen. The lack of intrasyngenic variation permits assessment of intersyngenic relationships. Syngens differ in a complex way from each other, suggesting that several gene differences may be involved. The syngens can be classified on the basis of their esterases. Syngens which have been shown to be more closely related in terms of cross-mating, breeding systems, and other criteria tend to be more similar in their esterase isozymes. The isozyme technique confirms relationships previously suggested among syngens and offers the promise of eventual assessment of evolutionary distances among syngens. However, establishment of these relationships will be clearer in the absence of bacteria.Supported by a Research Grant, GM-15879, from the National Institute of General Medical Sciences, U.S. Public Health Service     

Esterase Variations between the 14 Syngens of PARAMECIUM AURELIA under Axenic Growth

Under axenic growth all 14 syngens of Paramecium aurelia have 4 types of esterases. The three major types (A, B and cathodal C) vary independently in electrophoretic mobility among the syngens. Using these three esterases, stocks can be keyed to a syngen, except for the groupings 1-3-5 and 7-13. Using 5 other enzymes only syngens 1 and 5 cannot be distinguished. Most syngens differ from each other in 6 out of the 8 enzymes. An axenically-grown stock of Paramecium multimicronucleatum collected in Costa Rica has the same types of esterases as P. aurelia. Two of the types (A and C) are similar in mobility to those found in syngens 7 and 13, but its B esterase differs in mobility from all the known syngens of P. aurelia.     

Enhancement of anti-tumor immunity specific to murine glioma by vaccination with tumor cell lysate-pulsed dendritic cells engineered to produce interleukin-12

Aim: The aim of this study was to develop an immunotherapy specific to a malignant glioma by examining the efficacy of glioma tumor-specific cytotoxic T lymphocytes (CTL) as well as the anti-tumor immunity by vaccination with dendritic cells (DC) engineered to express murine IL-12 using adenovirus-mediated gene transfer and pulsed with a GL26 glioma cell lysate (AdVIL-12/DC+GL26) was investigated. Experimentl: For measuring CTL activity, splenocytes were harvested from the mice immunized with AdVIL-12/DC+GL26 and restimulated with syngeneic GL26 for 7 days. The frequencies of antigen-specific cytokine-secreting T cell were determined with mIFN-γ ELISPOT. The cytotoxicity of CTL was assessed in a standard 51Cr-release assay. For the protective study in the subcutaneous tumor model, the mice were vaccinated subcutaneously (s.c) with 1×10⁶ AdVIL-12/DC+GL26 in the right flanks on day −21, −14 and −7. On day 7, the mice were challenged with 1×10⁶ GL26 tumor cells in the shaved left flank. For a protective study in the intracranial tumor model, the mice were vaccinated with 1×10⁶ AdVIL-12/DC+GL26 s.c in the right flanks on days −21, −14 and −7. Fresh 1×10⁴ GL26 cells were inoculated into the brain on day 0. To prove a therapeutic benefit in established tumors, subcutaneous or intracranial GL26 tumor-bearing mice were vaccinated s.c with 1×10⁶ AdVIL-12/DC+GL26 on day 5, 12 and 19 after tumor cell inoculation. Results: Splenocytes from the mice vaccinated with the AdVIL-12/DC+GL26 showed enhanced induction of tumor-specific CTL and increased numbers of IFN-γ: secreting T cells by ELISPOT. Moreover, vaccination of AdVIL-12/DC+GL26 enhanced the induction of anti-tumor immunity in both the subcutaneous and intracranial tumor models. Conclusions: These preclinical model results suggest that DC engineered to express IL-12 and pulsed with a tumor lysate could be used in a possible immunotherapeutic strategy for malignant glioma.Korea Research Foundation Grant (KRF-2004-005-E00001).     

The Syngens of Paramecium bursaria: New Mating Types and Intersyngenic Mating Reactions

SYNOPSIS. Three syngens of Paramecium bursaria have been identified amongst stocks collected in Scotland. These syngens probably correspond to the previously-described syngens 4, 5 and 6; they have been so named. In all 3 syngens 8 mating types have been found. An extensive series of intersyngenic mating reactions has been discovered between stocks of syngens 4 and 5, and between stocks of syngen 2 and syngens 4 and 5.