Paramecium decaurelia and Paramecium dodecaurelia from the P. aurelia spp. complex in Japan

The presence of Paramecium decaurelia (three strains) and Paramecium dodecaurelia (two strains) were recorded in Japan, for the first time in this country and outside the USA.     

Paramecium biaurelia--relation of strains

Inter- and intra-strain crosses were made in Paramecium biaurelia of the P. aurelia species complex for studying the relation of strains within the species. Altogether ten strains originating from Scotland, Spain, Romania, Czech Republic, Ukraine, Italy, Germany, Russia, and Poland (two strains) were studied. A high percentage of surviving clones in both generations, F1 (obtained by conjugation) and F2 (obtained by autogamy), was observed in strain crosses, indicating a strong relation between the strains, and absence of genetic barriers between them in P. biaurelia.     

Nutritional requirements and biochemical activities of pineapple pink disease bacterial strains from Hawaii

Bacteria which cause pink disease of pineapple, identified on the basis of their nutritional and biochemical activities, were found to belong to three genera. These bacteria include the following species: Gluconobacter oxydans, Acetobacter aceti, and Erwinia herbicola. Several pink disease strains required one to three vitamins for growth. Both G. oxydans strains 303D and 180 required biotin, nicotinic acid, and pantothenic acid for growth; E. herbicola 189 required only nicotinic acid; however, A. aceti 295 was able to grow without any added supplements in glucose mineral salts medium. Optimal vitamin concentrations for maximal growth and optimal pH for the maximal number of generations per hour was established for a few pink disease strains.Journal Series Paper No. 2373 of the Hawaii Agricultural Experiment Station, Honolulu.     

Gnathostomulida from Hawaii

As thc second in a series on Gnathostomulida from the Pacific Ocean this paper describes an additional eight species from Hawaii of which four are new to science. All are accommodated within existing genera: Haplognathia asymmetrica sp.n., Haplognathia ruberrima (Sterrer, 1966), Haplognathia rufa sp.n., Cosmognathia arcus Sterrer, 1991, Cosmognathia manubrium sp.n., Pterognathia ctenifera Sterrer, 1970, Pterognathia hawaiiensis sp.n., and Austrognathia novaeze landiae Sterrer, 1991.     

Disparity of native oxyhemoglobin components isolated from Paramecium caudatum and Paramecium primaurelia

• 1.1. Native oxyhemoglobin components were isolated chromatographically from Paramecium caudatum and Paramecium primaurelia, and some properties of the isolated components were investigated.
• 2.2. P. caudatum was endowed with one homogeneous hemoglobin component, while the hemoglobin in P. primaurelia was resolved into three heterogeneous components being two main and one minor.
• 3.3. Spectral properties of the isolated hemoglobin components were quite similar to each other. The isolated components, however, were distinctly different in electrophoretic mobilities.
• 4.4. Molecular weight of the isolated hemoglobin components was estimated to be about 11,000.

     

Molecular polymorphism of strains within Paramecium septaurelia (Ciliophora, Oligohymenophorea)

RAPD-PCR fingerprinting and ARDRA riboprinting revealed polymorphism within P. septaurelia strains from Russia (4 strains from Lower Volga Basin), and one strain from USA, Florida. However, the first method showed the existence of four RAPD genotypes while the second revealed only two groups of strains with different band patterns. All studied strains had a high percentage of surviving hybrid clones in the inter-strain crosses, with little differentiation of strains within species. Intra-species differentiation of strains in RAPD band patterns may be connected with the degree of inbreeding for the studied species. Species of the P. aurelia complex can be arranged according to the degree of inbreeding characteristic for each, which is correlated with the degree of DNA polymorphism revealed by the RAPD method from extreme inbreeders (e.g. P. sexaurelia), moderate inbreeders (e.g. P. triaurelia) to weak inbreeders (e.g. P. pentaurelia). P. septaurelia of the P. aurelia complex should be included in the group of extreme inbreeder.     

Random amplified polymorphic DNA fingerprinting as a marker for Paramecium jenningsi strains

The aim of the present study is to establish a common RAPD marker for P. jenningsi using a series of Ro primers and to investigate if strains originating from distant and isolated localities (Japan, China, India, Saudi Arabia) have isolated gene pools and represent distinct species. An analysis of dendrograms constructed on the basis of RAPD-PCR fingerprints with four primers (Ro 460-04, 460-06, 460-07, and 460-10) from the first part of this project (SKOTARCZAK et al. 2004), assigns the strains to two groups consisting of the continental strains (India, Saudi Arabia, China) and Japanese strains that have been considered as a separate sibling species within P. jenningsi. The genetic similarity of the Indian and Arabian strains was ascertained, whereas the Chinese strain formed an independent branch in this sibling species. The primers Ro (460-01,460-02, 460-03, 460-05, 460-08) also distinguish between two groups of strains, although they divide the Japanese strains into two subgroups that are not reproductively isolated. This probably indicates genetic variation within this sibling species. However, it comprises one common gene pool (successful inter-strain crosses) and is reproductively isolated from the other sibling species. The results presented in these papers confirm that the construction of ten band patterns having marker attributes is possible on the basis of DNA amplification from 9 strains of P. jenningsi with the RAPD-PCR fingerprinting method using five primers from the Ro series. The patterns can be assigned to three marker-groups: a general species group, a group differentiating between sibling species, and accessory strain markers.     

Characteristics of emigrants from Hawaii

As part of an ongoing study on young adult psychological and social development, data were obtained through parental reports on the present residences and educational and occupational attainments of 718 present or former residents of Hawaii (average age 31 years). These subjects, as well as their parents, had been tested between 1972 and 1976 on measures of cognitive abilities and personality. The extent of emigration to the mainland in this middle to upper-middle class sample was over 40%. On average, former Hawaii residents now living on the US mainland were of higher intelligence and educational background than their counterparts still living in Hawaii. Differences were also found for number of children, cross-ethnic marriages, and occupational attainment (males only). In addition, parents of US mainland residents scored significantly higher on measures of cognitive abilities and education than parents of current Hawaii residents.     

Isolation of surface membranes from normal and exocytotic mutant strains of Paramecium tetraurelia. Ultrastructural and biochemical characterization

A density gradient centrifugation method for the isolation of the surface membrane complex from Paramecium tetraurelia cells is presented. The resulting "pellicles" consist predominantly of the somatic cell membrane and the underlying alveolar membranes. Marker enzyme activities for other cell components are low and SDS-polyacrylamid-gel electrophoreses indicate the presence of only minor amounts of ciliary and secretory proteins. Pellicles were prepared from different strains: (a) Exocytosis-capable strains with the normal set of exocytotic organelles ("trichocysts") docked to the cell membrane (strains 7S, K 401, and 9-18 degrees C), (b) exocytosis-uncapable strains (although with normal trichocyst attachment: nd 9-27 degrees C, nd 6, nd 7) and (c) strain from tam 38 with empty docking sites and rare, defective, free trichocysts. A Ca2+-stimulated ATPase was present in the pellicles from all strains with Km (CA2+) values between 0.19 to 0.88 mM Ca2+ and Vmax between 286 to 787 nMoles Pi/mg protein/min. Km and Vmax was identical for all strains of group (a). Vmax was significantly lower for all strains of group (b) and still lower for group (c). Similar group differences were found for Km (except for strain nd 6). Freeze-fracture analysis shows that the disruption of the membrane-to-membrane attachments during fractionation is paralleled by the disarrangment of the regular arrays ("rings", "rosettes") of membrane-integrated particles.     

Organization and closing of mitochondrial deoxyribonucleic acid from Paramecium tetraaurelia and Paramecium primaurelia.

Previously we showed that the mitochondrial deoxyribonucleic acid (DNA) from Paramecium aurelia consists of a linear genome and that replication of this genome is initiated at one terminus and proceeds unidirectionally to the other terminus. Analyses of mitochondria from four closely related species (1, 4, 5, and 7) indicated that the species 1, 5, and 7 DNAs are essentially completely homologous but that the species 4 mitochondrial DNA is only 40 to 50% homologous with that from species 1. The major regions of homology are those containing the genes for ribosomal ribonucleic acid (RNA). To understand the replication and organization of the linear mitochondrial genome better, we compared species 1 (Paramecium primaurelia) and 4 (Paramecium tetraaurelia) DNAs with regard to restriction fragment mapping and homology between initiation regions; we also identified the sites of the genes for ribosomal RNA. In general, the structures of the species 1 and 4 mitochondrial genomes were quite similar. Each ribosomal RNA gene was present in one copy per genome, with the large ribosomal RNA gene located near the terminal region of replication and the small ribosomal RNA gene located more centrally. These two genes were separated by about 10 kilobases in the species 1 genome and by about 12 kilobases in the species 4 genome. In contrast to our previous findings, by using nonstringent hybridization conditions we detected homology between the species 1 and 4 DNA fragments containing the initiation regions. We constructed recombinant DNA clones for many fragments, especially those containing the initiation region and the ribosomal RNA genes. We also constructed restriction enzyme maps for six enzymes for both P. primaurelia and P. tetraaurelia.     

Phylogenetic relationships of Paramecium jenningsi strains (classical analysis and RAPD studies)

The present studies with application of classical strain crosses and RAPD-PCR analyses showed the existence of different genetic species (syngens) within Paramecium jenningsi. So far the existence of only one syngen has been accepted. It was found that strains from Saudi Arabia, India, and China compose one genetic species (syngen 1) and six strains from Japan compose second genetic species (syngen 2).     

Photo-oxidative stress in symbiotic and aposymbiotic strains of the ciliate Paramecium bursaria.

We tested the hypothesis that photo-oxidative stress is greater in symbiotic representatives of the freshwater ciliate Paramecium bursaria than in aposymbiotic (i.e., without Chlorella) ones. The level of oxidative stress was determined by assessing reactive oxygen species (ROS) with two fluorescent probes (hydroethidine and dihydrorhodamine123) by flow cytometry in exponential and stationary growth phases of both strains. Photo-oxidative stress was assessed in the laboratory after exposure of the ciliates to photosynthetically active radiation (PAR: 400-700 nm) and PAR+ultraviolet radiation (UVR: 280-400 nm). Additionally, both strains were screened for their antioxidant defenses by measuring the activity of the enzymes catalase, superoxide dismutase (SOD), and glutathione reductase. The results showed that aposymbiotic ciliates had higher levels of PAR-induced oxidative stress than symbiotic ones. Significant differences in PAR-induced oxidative stress were also found in both strains when comparing exponential and stationary growth phases with generally higher values in the former. After exposure to UVR, aposymbiotic ciliates in the stationary phase had the highest levels of ROS despite an increase in SOD activity. By contrast, exposure to UVR decreased catalase activity in both strains. Overall, our results suggest that in this ciliate symbiosis, the presence of symbionts minimizes photo-oxidative stress. This work represents the first assessment of photo-oxidative stress in an algal-ciliate mutualistic symbiosis.     

Comparative studies on photosynthetic enzymes of the symbiotic Chlorella from Paramecium bursaria and other symbiotic and non-symbiotic Chlorella strains

The activities of ribulose 1,5-bisphosphate carboxylase and of carbonic anhydrase were studied in cell-free extracts of two symbiotic Chlorella strains isolated from Paramecium bursaria and from Spongilla sp., and of two nonsymbiotic strains of Chlorella (Chlorella fusca and Chlorella vulgaris) cultivated at varied CO₂-concentrations. The symbiotic Chlorella of Paramecium bursaria differs distinctly from the other Chlorella strains by a higher activity of ribulose 1,5-bisphosphate carboxylase, which is independent of the actual CO₂-concentration, and by a lack of carbonic anhydrase activity. These properties are discussed with respect to their ecological significance.Abbreviations CA carbonic anhydrase - Pbi Paramecium bursaria isolate - RuBP ribulose 1,5-bisphosphate Dedicated to Prof. Dr. André Pirson on the occasion of his 70th birthday     

Structure of mitochondrial DNA from Paramecium tetraurelia

Mitochondrial DNA (mtDNA) from endosymbiote-free stocks of Paramecium tetraurelia was isolated by 2 procedures. The buoyant density of the mtDNA in neutral CsCl was 1.702 gm/cm3, a value consistent with the melting temperature of the mtDNA. Only linear molecules were observed by electron microscopy. These molecules were homogeneous in size with a monomer molecular weight of 25.6 x 10(6) daltons. The size of the mtDNA determined after digestion with the restriction endonucleases EcoRI or Hind III agreed with the value obtained by electron microscopy. These studies also revealed that the digestion pattern of mtDNA from stock 172 differed from that of other 3 stocks (51, 127, 203) examined. Some mtDNA molecules exhibited snapback reassociation following denaturation.     

Anatomy of mitochondrial DNA from Paramecium aurelia

Summary The linear genome of mitochondrial DNA from four species of Paramecium aurelia was investigated with respect to restriction endonuclease fragments, location and number of ribosomal RNA genes, and interspecies EcoRI and HindIII fragment homologies. One copy of each of the rRNA genes was found in all four species and the 14s and 20s rRNA genes were separated by at least 3,000 bp. R-Loop analysis of the 20s rRNA gene did not reveal the presence of an intervening sequence. Interspecies homology studies showed species 1, 5, and 7 to have a high degree of homology but species 4 was less than 50% homologous to species 1 mt DNA. For all four species, rRNA genes showed good homology indicating that these DNA sequences are highly conserved, even between species having many non-homologous regions. A major region of DNA which displayed little homology between species 1 and 4 was that fragment containing sequences essential for initiation of DNA replication.     

A novel cGMP-dependent protein kinase from Paramecium

An unusual monomeric cGMP-dependent protein kinase, enriched in cilia, was isolated from Paramecium cilia and whole cells. Cilia and whole cell extracts had relatively high ratios of cGMP-dependent to cAMP-dependent protein kinase activity (1:2). The calculated molecular weight of the native enzyme was 88,000. The enzyme was identified on sodium dodecyl sulfate-polyacrylamide gels as a 77,000 molecular weight band based on copurification of this protein with enzyme activity, 8-N3-[32P]cAMP labeling, and autophosphorylation. Based on the size of the native enzyme, it was concluded that the kinase is a monomer with cGMP-binding and catalytic activities on the same polypeptide. Dimer-sized cGMP-dependent protein kinase, like that of the well characterized mammalian enzyme, was never seen, despite stringent efforts to control proteolysis. The structure of the Paramecium cGMP-dependent protein kinase supports a model in which the dimeric vertebrate form of the enzyme evolved from an early monomeric form. The catalytic properties of the Paramecium enzyme differed in several respects from those of the mammalian enzyme: it could use GTP or ATP as the phosphoryl donor, it did not phosphorylate Kemptide effectively, and it had poor histone kinase activity with high Mg2+ concentrations. Quercertin, 5'-guanylyl imidodiphosphate, indomethacin, and the isoquinolinesulfonamide drug H7 inhibited Paramecium cGMP-dependent protein kinase activity. The enzyme had fast and slow binding sites (with kd values of 5-10 x 10(-3)s-1 and 0.44 x 10(-3)s-1) and showed an order of preference for cyclic nucleotides and cyclic nucleotide analogs similar to that of the mammalian enzyme.