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.     

High coding density on the largest Paramecium tetraurelia somatic chromosome

Paramecium, like other ciliates, remodels its entire germline genome at each sexual generation to produce a somatic genome stripped of transposons and other multicopy elements. The germline chromosomes are fragmented by a DNA elimination process that targets heterochromatin to give a reproducible set of some 200 linear molecules 50 kb to 1 Mb in size. These chromosomes are maintained at a ploidy of 800n in the somatic macronucleus and assure all gene expression. We isolated and sequenced the largest megabase somatic chromosome in order to explore its organization and gene content. The AT-rich (72%) chromosome is compact, with very small introns (average size 25 nt), short intergenic regions (median size 202 nt), and a coding density of at least 74%, higher than that reported for budding yeast (70%) or any other free-living eukaryote. Similarity to known proteins could be detected for 57% of the 460 potential protein coding genes. Thirty-two of the proteins are shared with vertebrates but absent from yeast, consistent with the morphogenetic complexity of Paramecium, a long-standing model for differentiated functions shared with metazoans but often absent from simpler eukaryotes. Extrapolation to the whole genome suggests that Paramecium has at least 30,000 genes.     

Ultrastructure of the proximal region of somatic cilia in Paramecium tetraurelia

The morphology of the transition zone between the terminal plate of the basal body and the 9 + 2 region of the somatic (non-oral) cilium has been examined in Paramecium tetraurelia. Freeze-fracture and thin- section techniques disclosed both membrane specializations and various internal structural linkages. Freeze-fracture material revealed sets of particles interrupting the unit membrane. The more distal of these form plaquelike arrays while the proximal set of particles forms the ciliary "necklace." The plaque regions correspond to anionic sites on the outer membrane surface as revealed by binding of polycationic ferritin. Both the plaque particles and the necklace particles appear to be in contact with outer doublet microtubules via a complex of connecting structures. In the interior of the transition zone an axosomal plate supports an axosome surrounded by a ring of lightly packed material. Only one of the two central tubules of the axoneme reaches and penetrates the axosome. Below the axosomal plate four rings, each approx. 20 nm wide, connect adjacent outer doublets. An intermediate plate lies proximal to these rings, and a terminal plate marks the proximal boundary of this zone. Nine transitional fibers extend from the region of the terminal plate to the plasmalemma. The observations described above have been used to construct a three-dimensional model of the transition region of "wild-type" Paramecium somatic cilia. It is anticipated that this model will be useful in future studies concerning possible function of transition-zone specializations, since Paramecium may be examined in both normal and reversed ciliary beating modes, and since mutants incapable of reverse beating are available.     

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.     

Control of DNA excision efficiency in Paramecium

Programmed excision of internal eliminated sequences (IESs) occurs at thousands of sites in ciliate genomes. How this is controlled is largely unknown. Here, we report the characterization of the non-efficiently excised 156ψG-11 IES from Paramecium primaurelia strain 156 and that of the efficiently excised 168ψG-11 IES, an allelic variant from strain 168. Then, we report a genetic and molecular analysis of IES excision efficiency in F₁ progeny derived from interstrain crosses and in F₂ homozygous progeny derived from F₁ autogamy. IES 168ψG-11 excision efficiency was ~100% in all cases. IES 156ψG-11 excision efficiency was 19 ± 13% in F₁ progeny and 0.6 ± 1.1% in F₂ progeny. No trans-excision event between IESs 156ψG-11 and 168ψG-11 was detected within the F₁ progeny. These data demonstrate that the excision efficiency of this IES is variable and controlled by a cis-acting element. This should encompass positions 8 and/or 9 of the right IES end, which display allele differences. Finally, the 30-fold stimulation of IES 156ψG-11 excision efficiency within F₁ progeny relative to F₂ progeny demonstrates that Paramecium IES excision efficiency is sensitive either to a conjugation-specific trans-acting factor provided by the zygotic genome, or to homologous chromosome cross-talk.     

Veratridine triggers exocytosis in Paramecium cells by activating somatic Ca channels

Paramecium tetraurelia wild-type (7S) cells respond to 2.5 mm veratridine by immediate trichocyst exocytosis, provided [Ca²⁺] _o (extracellular Ca²⁺ concentration) is between about 10^–4 to 10^–3 m as in the culture medium. Exocytosis was analyzed by light scattering, light and electron microscopy following quenched-flow/ freeze-fracture analysis. Defined time-dependent stages occurred, i.e., from focal (10 nm) membrane fusion to resealing, all within 1 sec.Veratridine triggers exocytosis also with deciliated 7S cells and with pawn mutants (without functional ciliary Ca channels). Both chelation of Ca²⁺ _o or increasing [Ca²⁺] _o to 10^–2 m inhibit exocytotic membrane fusion. Veratridine does not release Ca²⁺ from isolated storage compartments and it is inefficient when microinjected. Substitution of Na⁺ _o for N-methylglucamine does not inhibit the trigger effect of veratridine which also cannot be mimicked by aconitine or batrachotoxin. We conclude that, in Paramecium cells, veratridine activates Ca channels (sensitive to high [Ca²⁺] _o ) in the somatic, i.e., nonciliary cell membrane and that a Ca²⁺ influx triggers exocytotic membrane fusion. The type of Ca channels involved remains to be established.We thank Dr. C. Kung (Madison, WI) for providing the pawn mutant, Drs. G. Lehle and R. Waldschütz-Schüppel (Konstanz, Germany) for their help with light scattering experiments, and Ms. E. Dassler and D. Bliestle for continuous help during the extensive photographic documentation. This work has been supported by Deutsche Forschungsgemeinschaft, Schwerpunkt Neue mikroskopische Techniken für Biologie und Medizin (grant P178/11) and SFB156/B4.     

Specific-primer-directed DNA sequencing

A simple and rapid strategy for DNA sequence analysis based on the Sanger chain-termination method is described. This procedure utilizes full-sized inserts of 1 to 4 kb of DNA cloned into M13 bacteriophage vectors. After the sequence of the first 600-650 bp of the insert DNA has been determined with the commercially available universal vector primer, a specific oligonucleotide is synthesized utilizing the sequence data obtained from the 3' end of the sequence and used as a primer to extend the sequence analysis for another 600-650 nucleotides. Additional primers are synthesized in a similar manner until the nucleotide sequence of the entire insert DNA has been determined. General guidelines for the selection of oligonucleotide length and composition and the use of unpurified primers are discussed. The use of the specific-primer-directed approach to dideoxynucleotide sequence analysis, in association with highly purified single-stranded template DNA, reduces considerably the time required for the analysis of large segments of DNA.     

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.     

Mass-spectrometry DNA sequencing

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been explored widely for DNA sequencing. Compared to gel electrophoresis based sequencing systems, mass spectrometry produces very high resolution of sequencing fragments, rapid separation on microsecond time scales, and completely eliminates compressions associated with gel-based systems. While most of the research efforts have focused on using mass spectrometers to analyze the DNA products from Sanger sequencing or enzymatic digestion reactions, the read lengths attainable are currently insufficient for large-scale de novo sequencing. The advantage of mass-spectrometry sequencing is that one can unambiguously identify frameshift mutations and heterozygous mutations making it an ideal choice for resequencing projects. In these applications, DNA sequencing fragments that are the same length but with different base compositions are generated, which are challenging to consistently distinguish in gel-based sequencing systems. In contrast, MALDI-TOF MS produces mass spectra of these DNA sequencing fragments with nearly digital resolution, allowing accurate determination of the mixed bases. For these reasons mass spectrometry based sequencing has mainly been focused on the detection of frameshift mutations and single nucleotide polymorphisms (SNPs). More recently, assays have been developed to indirectly sequence DNA by first converting it into RNA. These assays take advantage of the increased resolution and detection ability of MALDI-TOF MS for RNA.     

5-Azacytidine affects the programming of expression of the somatic nucleus of Paramecium

This report introduces a new system in the study of programming of genomic function during development of the somatic nucleus of Paramecium tetraurelia. Previous works have established a definite, but replaceable, role of the germ nuclei (micronuclei) in oral development in the asexual cycle; their removal from the cell generates viable amicronucleate cell lines, which characteristically suffer a transient period of growth depression marked by abnormal oral development. Such cell lines gradually recover, showing that a compensatory mechanism is activated in the absence of the germ nuclei to bring the cell back to near-normal. To test the notion that the somatic nucleus (macronucleus) is involved in this compensation, cells possessing micronuclei were treated with 5-azacytidine during sexual reproduction when new somatic nuclei develop. These cells were then propagated asexually for a number of fissions in the absence of the drug, and thereafter micronuclei were removed from them. The amicronucleate cell lines generated in this manner clearly did not suffer a depression as severe as the untreated controls did in terms of growth rate and oral development, and they recovered much sooner. This supports the notion that the somatic nucleus is the physical basis of the compensatory mechanism. This study suggests that the stomatogenic sequences in question normally become repressed in the somatic nucleus developing in sexual reproduction, and that 5-azacytidine administered to the cells at this time could alter this programme which then persists during subsequent asexual propagation. The possibility that the somatic nucleus is programmed by methylation of cytosine at the 5' position is discussed.     

Accumulation of DNA damages in aging Paramecium tetraurelia

Summary Paramecium tetraurelia cells of ages 4, 15, and 27 days were labeled with [¹⁴C]-thymidine. In addition, cells were grown clonally for 27 days (108 generations) and labeled with [¹⁴C]-thymidine in the presence of 0.5 or 7.5 g/ml of mitomycin-C (MMC) or no MMC. These cells were gently deposited on a filter membrane, which impedes the passage of DNA strands. The cells were then lysed with detergents and the cellular components washed through the filters, leaving double-stranded DNA intact on the surface. Proteinase K was used to remove histone or DNA-bound proteins. The DNA was then eluted under alkaline conditions, which denatures double-stranded DNA and converts apurinic/apyrimidinic sites into single-strand breaks. The results obtained with the cells of ages 4, 15, and 27 days (16, 60, and 108 generations, respectively) indicate that as Paramecium tetraurelia ages during asexual reproduction, apurinic/apyrimidinic lesions, strand breaks or single-strand gaps accumulate. This accumulation may be the basic mechanism of aging in such cells. In the MMC-treated cells of 27 days (108 generations), the MMC reduced elution of DNA fragments more at the higher than at the lower pH's used; random MMC cross-links should occur more often in longer strands than in shorter strands. The reductions in elution preferentially at higher pH, at which longer single strands would be eluted, confirmed the pH-versuslength relationship for Paramecium DNA eluted under our conditions.     

DNA amounts in the nuclei of Paramecium bursaria

DNA amounts in the macronuclei and micronuclei of syngen 4 of Paramecium bursaria were determined. There was a wide distribution of values for the amount of DNA in the macronucleus. This distribution remained wide, but moved to lower values as the animals were starved. The micro-nuclear DNA values were constant under all culture conditions, at a value of 7.5 × 10^–12 g per nucleus. This gave an estimate of the haploid genome size, 2.25 × 10¹² daltons, which is approximately ten times that obtained by DNA renaturation studies.This work was supported by a Commonwealth Scholarship to the author. Thanks are due to the Medical Research Council for funds to purchase the Vickers densitometer.     

Colorimetric-detected DNA sequencing

A sensitive, colorimetric method for visualizing the band pattern of DNA sequencing reaction is described. The enzymatic incorporation of radioactive nucleotides commonly used for the band detection is replaced by biotin conjugated to the 5'-terminus of a synthetic oligonucleotide by chemical synthesis. The oligonucleotide so labeled is used as a primer for dideoxy DNA sequencing in a primer extension reaction. The products of the sequencing reactions are analyzed on a denaturing polyacrylamide gel using the direct blotting electrophoresis technique. This technique makes it possible to transfer the band pattern during the electrophoresis onto an immobilizing matrix, on which it is made visible by an enzymatic reaction in less than 3 h. This biotin-based detection method is so sensitive that the sequencing reactions can be performed under the same conditions and concentrations as those for the radioactive detection.     

Regulation of macronuclear DNA content in Paramecium tetraurelia

The macronucleus of Paramecium divides amitotically, and daughter macronuclei with different DNA contents are frequently produced. If no regulatory mechanism were present, the variance of macronuclear DNA content would increase continuously. Analysis of variance within cell lines shows that macronuclear DNA content is regulated so that a constant variance is maintained from one cell generation to the next. Variation in macronuclear DNA content is removed from the cell population by the regulatory mechanism at the same rate at which it is introduced through inequality of macronuclear division. Half of the variation in macronuclear DNA content introduced into the population at a particular fission by inequality of division is compensated for during the subsequent period of DNA synthesis. Half of the remaining variation is removed during each subsequent cell cycle. The amount of variation removed in one cell cycle is proportional to the postfission variation. The cell's power to regulate DNA content is substantially greater than that required to compensate for the small differences that arise during division of wild-type cells. For example, a constant variance was still maintained when the mean difference between sister cells was increased to ten times its normal level in a mutant strain. The observations are consistent with a replication model that assumes that each cell synthesizes an approximately constant amount of DNA which is independent of the initial DNA content of the macronucleus. It is suggested that the amount of DNA synthesized may be largely determined by the mass of the cell.     

Evolutionary divergence of mitochondrial DNA from Paramecium aurelia

Summary Mitochondrial (mt) DNA from four sibling species within the Paramecium aurelia complex, including stocks of different geographic origin and mutants, were analyzed using four 6-bp recognition site and one 4-bp recognition site endonucleases and the sequence divergence was estimated using Upholt's (1977) statistical procedure. All four species were readily distinguishable regardless of the restriction endonuclease employed. With intraspecies comparisons, no differences were observed which could be accounted for on the basis of geographic origin. Except for species 4, each stock (and mutant) gave a species-specific fragment pattern. For species 4, while the patterns were distinct from the other species, two species-specific type of patterns were found, designated A and B. The sequence divergence between these was estimated to be between 1 and 2 percent. With interspecies comparisons, the sequence divergence ranged from 3.9 to 10.3% with the greatest divergence being between species 1 and 4, and the least between species 1 and 5. The similarity between species 1 and 5 is in accord with other criteria for interspecies comparisons. The degree of sequence divergence measured here in Paramecium mt DNA is well within the range reported for rodents and primates. All four species mt DNA were cleaved to many DNA fragments by DPN II, an enzyme which recognizes non-methylated sites, and not by DPNI, the methyl-site specific counterpart of DPN II, suggesting that mt DNA from Paramecium aurelia is not appreciably methylated, if at all.