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PubMed1992

The beta-tubulin genes of Paramecium are interrupted by two 27 bp introns.

In the ciliate Paramecium tetraurelia, the analysis of the tubulin gene family has revealed the existence of four alpha and three beta genes. We show here that the coding sequence of the first beta-tubulin gene to be cloned and sequenced is interrupted by two short non-coding sequences of 27 bp each, which present at their extremities the pairs GT/AG, characteristic of eukaryotic pre-mRNA introns, and the internal pentanucleotide TTAAT, consensual in Tetrahymena introns. We demonstrate by PCR experiments that the three macronuclear beta-tubulin genes contain these sequences in similar positions, thereby ruling out the possibility that these sequences are ciliate IES present in the micronucleus and eliminated in the transcriptionally active macronucleus. S1 mapping analysis and mRNA sequencing show that the sequences are absent from the beta-tubulin transcripts. These sequences are the first introns described in protein encoding genes in P. tetraurelia and the shortest known introns altogether.

Amino Acid Sequence↗
PubMed1993

Developmental precise excision of Oxytricha trifallax telomere-bearing elements and formation of circles closed by a copy of the flanking target duplication.

The 4.1 kbp TBE1 elements of Oxytricha fallax and Oxytricha trifallax are deduced to transpose into a centrisymmetric target, CAnTG, and to duplicate the central AnT. Despite conserved C(A4C4)2 telomeric repeats at their tips, free TBE1s found during macronuclear development are not linear but 4.1 kbp circles closed on one copy of the AnT target duplication. The macronucleus-destined flanks are rejoined to regenerate the target, effecting efficient and precise somatic reversion of the germline transpositional mutation. A model is presented in which transposase catalyzes concerted precise rejoining of the flanks and cyclization of the excised element.

Animals↗
PubMed1987

Longevity in the protozoa.

In ciliates there are examples of cells which have different proliferation potential in the macronucleus. Those species with limited macronuclear proliferation potential require sex to activate the reserve nucleus. In terms of the capital investment theory, some ciliates invested in their spare nucleus without loss of their original potential, while others accumulated debts and needed the reserve account to maintain life. Other cells neglected maintenance of their reserve account and failed unless their venture capital account was not a self-sustaining venture. Sex provided access to the reserve account and had to occur before deterioration of the reserve account. The question is not when cellular immortality was lost, but rather when immortality was partitioned from a mortal segment. The separation provided the option both for senescence and evolution in multicellular organisms. In colonial flagellates, separation of cells with infinite and finite cell lifespan potential occurred in some species, while in others the separation did not involve loss of immortality. In colonial flagellates, sex did not become an obligate stage. The immortal cells are haploid and could not accumulate damage and live (in contrast with the diploids in the ciliated protozoans). The present theory predicts that differences between species or cells with infinite versus finite lifespan potential may reveal differences in the critical determinants of longevity. Senescence could arise as an accident, as well as a design of nuclear differentiation. Cells therefore may have a much greater reserve for totipotency than would be predicted if they were assumed to lose immortality simply by the act of differentiation.

Aging↗
PubMed1984

Elimination of germ-line tandemly repeated sequences from the somatic genome of the ciliate Oxytricha fallax.

The ciliated protozoa exhibit nuclear dimorphism. The genome of the somatic macronucleus arises from the germ-line genome of the micronucleus following conjugation. We have studied the fates of highly repetitious sequences in this process. Two cloned, tandemly repeated sequences from the micronucleus of Oxytricha fallax were used as probes in hybridizations to micronuclear and macronuclear DNA. The results of these experiments show: (1) the cloned repeats are members of two apparently unrelated repetitious sequence families, which each appear to comprise a few percent of the micronuclear genome, and (2) the amount of either family in the macronuclei from which our DNA was prepared is about 1/15 that found in an equal number of diploid micronuclei. Most, if not all, of the apparent macronuclear copies of these repeats can be accounted for by micronuclear contamination, which strongly suggests that these sequences are eliminated from the macronuclei and have no vegetative function.

Cell Nucleus↗
PubMed1978

Free genes for rRNAs in the macronuclear genome of the ciliate Stylonychia mytilus.

When separated on an agarose gel, macronuclear DNA of the hypotrichous ciliate Stylonychia mytilus gives rise to many well-defined bands ranging in molecular weight from 0.3 x 10(6) to 14 x 10(6) dalton. Hybridization of 25 S rRNA, 17S rRNA or 5 S RNA to such a gel revealed sharp hybridization bands. This suggests that this banding pattern is not an artefact due to nonspecific degradation of macronuclear DNA but that the DNA in the macronucleus of Stylonychia occurs in discrete fragments, each coding for at least one gene. The size of the DNA fragment coding for rRNA was found to be 4.5 x 10(6) dalton, the fragment coding for 5 S RNA has a molecular weight of 150,000-250,000 dalton.

Animals↗
PubMed1981

Macronuclear chromatin organization in Paramecium primaurelia.

Chromatin organization and transcriptional activity in the macronucleus of the ciliate Paramecium primaurelia have been studied by electron microscopic examination of spread chromatin preparations. Under spreading conditions which inhibit the activity of endogenous nucleases and proteases, various morphologically distinct types of chromatin are seen: (1) Largely condensed chromatin in the form of 30-40 nm supranucleosomal globules which aggregate to form knobbly 40-60 nm fibres and unravel into 12 nm nucleosomal filaments; (2) In rapidly growing cells, extended chromatin arranged groups of 4-8 adjacent nucleofilaments, each bearing numerous and very densely packed lateral fibrils. These fibrils are 20 nm thick, have a smooth outline and extend to lengths of up to 3 micron. Such transcripts are grouped in units along the chromatin axis and are apparently organized in register in adjacent filaments; (3) In starved cells, extended nucleofilaments bearing only occasional RNP fibrils. These various morphological characteristics are discussed in terms of macronuclear transcriptional activity and the polyploid nature of macronuclei.

Animals↗
PubMed1980

Macronuclear DNA organization and transcription in Paramecium primaurelia.

Macronuclear DNA was isolated from Paramecium primaurelia, stock 168. Although the macronucleus is polyploid to the extent of 840C, in other respect the DNA appears to be simply organized, having neither satellite sequences nor substantial amounts of intermediately repetitive sequence. The sequence complexity of macronuclear DNA is quite low for a eukaryote cell, being approximately 19 times more complex than the genome of Escherichia coli. In addition, the GC content is low (25%) and the isolated DNA molecules have lengths mostly in the range 0.2-5 micrometer. In these various respects, the macronuclear DNA of Paramecium is similar to that of other ciliates. A clone of Paramecium cultured under controlled conditions contains polyadenylated RNA sequences which are homologous to 5-8% of the macronuclear DNA. Sequence complexity analysis indicates that the polyadenylated RNA contains two abundance classes of molecules, one present at low frequency and transcribed from approximately 10(4) genes, the other at 100 times greater concentration and transcribed from about 100 genes. The relevance of these results to the control of gene expression in Paramecium is discussed.

Animals↗
PubMed1975

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.

Animals↗
PubMed1980

Arrangement of coding and non-coding sequences in the DNA molecules coding for rRNAs in Oxytricha sp. DNA of ciliated protozoa. VII.

All of the genes in the macronucleus of Oxytricha sp. occur on physically separate "gene-sized" DNA molecules. We have inserted the DNA molecule that codes for rRNA into a bacterial plasmid in order to study its structure and function. Using restriction nuclease mapping and hybridization of 125I-rRNAs to gel separated DNA fragments we have determined that the intact rDNA is 8,140+/-50 base pairs (bp) in length. Reading from one end, the molecule consists of approximately 1,540 bp of non-coding DNA, approximately 2,100+/-50 bp that code for 19S rRNA, approximately 3,700+/-50 bp that code for 25 rTNA, and approximately 620+/-50 bp of non-coding DNDA. The 5.8S rRNA coding sequence (approximately 150 bp) occurs at one end of the 25S RNA coding region but which end is not known yet. All three rRNAs are encoded in the same strand of the DNA molecule, and transcription is in the order: 19S leads to 25S.

Animals↗
PubMed1978

Genome structure of Tetrahymena pyriformis.

Reassociation kinetics of DNA from the macronucleus of the ciliate, Tetrahymena pyriformis GL, has been studied. The genome size determined by the kinetic complexity of DNA was found to be 2.0 X 10(8) base pairs (or 1.2 X 10(11) daltons). About 90% of the macronuclear DNA fragments 200-300 nucleotides in length reassociate at a rate corresponding to single-copy nucleotide sequences, and 7-9% at a rate corresponding to moderate repetitive sequences; 3-4% of such DNA fragments reassociate at C0t practically equal to zero. To investigate the linear distribution of repetitive sequences, DNA fragments of high molecular weight were reassociated and reassociation products were treated with S1-nuclease. DNA double-stranded fragments were then fractionated by size. It has been established that in the Tetrahymena genome long regions containing more than 2000 nucleotides make up about half of the DNA repetitive sequences. Another half of the DNA repetitive sequences (short DNA regions about 200-300 nucleotides long) intersperse with single-copy sequences about 1,000 nucleotides long. Thus, no more than 15% of the Tetrahymena genome is patterned on the principle of interspersing single-copy and short repetitive sequences. Most of the so called "zero time binding" or "foldback" DNA seem to be represented by inverted self-complementary (palindromic) nucleotide sequences. The conclusion has been drawn from the analysis of this fraction isolated preparatively by chromatography. About 75% of the foldback DNA is resistant to S1-nuclease treatment. The S1-nuclease resistance is independent of the original DNA concentration. Heat denaturation and renaturation are reversible and show both hyper- and hypochromic effects. The majority of the inverted sequences are unique and about 20% are repeated tens of times. According to the equilibrium distribution in CsCl density gradients the average nucleotide content of the palindromic fraction does not differ significantly from that of total macronuclear DNA. It was shown that the largest part of this fraction of the Tetrahymena genome are not fragments of ribosomal genes.

Animals↗
PubMed1978

Histone genes in macronuclear DNA of the ciliate Stylonychia mytilus.

DNA in the macronucleus of Stylonychia mytilus exists as discrete gene-sized fragments which are derived from micronuclear DNA through a series of well-defined developmental events. It has been proposed that each of the DNA fragments might represent a gene and its controlling elements. We have investigated this possibility using genes which code for the five histone proteins. Macronuclear DNA fragments were fractionated according to size by agarose gel electrophoresis, the fragments transferred to nitrocellulose filters using the technique of Southern, and the filter-bound DNA hybridized with labeled cloned histone genes of the sea urchin, Psammechinus miliaris. Results indicate, first, that sequences homologous to the five individual histone gene probes are present in discrete macronuclear fragments which appear as bands in the gel hybridization assay. Secondly, for each of the five individual histone gene probes the homologous DNA fragments are several in number, ranging in size in from 7.6 Kb (Kilo base pairs) to 0.73 Kb. For example, the largest of six detected fragments hybridizing to the H3 gene probe contains approximately 10 times the amount of DNA required to code for a Stylonychia H3 histone. The smallest detected fragment hybridizing to the H3 probe contains enought DNA to code for approximately two copies of the histone. Finally, in general, no two histone approximately two copies of the histone. Finally, in general, no two histone gene probes hybridized to the same macronuclear DNA fragment. This result indicates that genes coding for the five histones in Stylonychia are not located together on the same macronuclear DNA fragments and implies that the five functionally related genes would not be transcribed together as a polycistronic unit.

Animals↗
PubMed1991

Structure of the macronuclear polyubiquitin gene in Euplotes.

The hypotrichous ciliate, Euplotes eurystomus, contains both a transcriptionally inactive micronucleus (MIC) and a transcriptionally active macronucleus (MAC) in the same cell. MAC DNA is small (0.5-20 kb), linear and highly amplified. Each DNA fragment consists of two telomeres, a single coding region, and the necessary control elements to regulate gene transcription and replication. The polyubiquitin gene consists of 898 bp, plus 28 bp of double-stranded and 14 bases of single-stranded DNA of the telomeric repeat G4T4 at each end. The coding region exists as three copies of the ubiquitin gene (690 bp) fused in a head-to-tail arrangement as in other organisms. The stop codon is TAA, as in other Euplotes genes, and is not the rare glutamine codon used in most other ciliates. The 3' nontranslated region contains two presumptive poly(A) addition sites; the 5' nontranslated region possesses two putative TATA boxes, several imperfect direct and inverted repeats, and a possible origin of replication. Nucleosome positioning studies reveal four tightly packed nucleosomes and a non-nucleosomal area containing the probable 5' control region as well as part of the coding region. The 5' area does not contain any DNAse I hypersensitive sites. Although the telomeres are protected from exonuclease digestion, they are not as well protected as Oxytricha telomeres against endonucleases and cleavage by methidium propyl Fe2+ EDTA.

Amino Acid Sequence↗
PubMed1985

Eliminated sequences with different copy numbers clustered in the micronuclear genome of Tetrahymena thermophila.

As the ciliated protozoan Tetrahymena thermophila develops a new macronucleus (MAC) from products of its micronucleus (MIC), several repetitive sequences are eliminated from the MAC genome. Four MIC DNA clones containing repetitive sequences that are eliminated from the MAC were obtained. One clone contains a representative from each of three families of eliminated sequences. One, present in 200-300 copies in the MIC, is almost completely eliminated from the MAC. A second, present in approximately 50 copies in the MIC, is scattered throughout the genome, although up to half of the family members examined could be localized to chromosome 2. Approximately one tenth of the members of this less repetitive family persist in the MAC while the rest are eliminated. The third type of eliminated sequence has three to four members, all of which are eliminated from the MAC. Three of the members are located on three of the five MIC chromosomes, and one could not be mapped. This sequence is clustered with the other two families of sequences in at least three of the four sites. All three types of eliminated sequences are found in similar arrangements in the MIC of several different inbred strains of T. thermophila.

Animals↗
PubMed1992

Properties of isolated extrachromosomal nucleoli from Tetrahymena pyriformis.

Extrachromosomal nucleoli were isolated from log phase cells of Tetrahymena pyriformis (amicronucleate strain) in a highly purified state. Nucleoli located at the periphery of the macronucleus were detached from the nucleoplasmic mass of isolated macronuclei with agitation and separated from macronuclei by filtration through a Nuclepore membrane filter (pore size 5 microm). The filtrate constitutes the crude nucleolar preparation, as judged by electron microscopy and DNA analysis. Further purification of the nucleoli was performed by isopycnic centrifugation of the filtrate in a Metrizamide density gradient. After this step, the purity of the nucleoli, as defined by rDNA content and measured by analytical CsCl centrifugation, was almost 100%. Electron microscopy of the purified nucleoli revealed structures that resemble those of an in situ nucleoli. Undegraded 35S pre-rRNA, together with 26S and 17S rRNA, could be isolated from purified nucleoli. In vitro RNA synthetic activity was associated with isolated nucleoli. This activity is insensitive to low and high concentrations of alpha-amanitin, indicating that the form I RNA polymerase is functioning.

Animals↗
PubMed1993

Inaccessibility of the Euplotes telomere binding protein.

The telomere binding protein (TP) from the macronucleus of the ciliate Euplotes eurystomus was purified by removal of tenaciously bound DNA with hydroxylapatite, and the purified TP partially sequenced. Rabbit antiserum was generated against a synthetic peptide of 14 amino acids at the amino-terminus of the TP. This antiserum was employed to examine the accessibility of TP antigenic determinants in nuclei and chromatin. Immunofluorescent staining of isolated macronuclei revealed only weak reactivity with specific antiserum. Reactivity within replication bands was demonstrated, and could be augmented by preparation of nuclear scaffolds. Employing a dot immunoblot analysis, the amino-terminal antigenic determinants of TP were revealed after extraction of histone H1 (and some nonhistones). A different aspect of TP inaccessibility was demonstrated by immunoblot analysis of trypsin-treated macronuclei and chromatin; TP was considerably less susceptible to digestion by trypsin than were histones H1 and H3. The relative inaccessibility of TP was not a consequence of chromatin higher-order structure, since soluble macronuclear chromatin in low salt exhibited the same burying of antigenic determinants by dot blot analysis, and the same decreased susceptibility to trypsin, as did isolated nuclei. Electron microscopy of soluble macronuclear chromatin spread in low salt revealed that most telomeres appear unfolded, without stable higher-order structure. The mechanisms for the relative inaccessibility of TP are not yet known, but probably arise as a consequence of the strong interactions of TP with the telomere nucleotide sequence and additional interactions of TP with various chromatin proteins, perhaps including histone H1.

Amino Acid Sequence↗
PubMed1992

Treatment of fish parasites. 9. Effects of a medicated food containing malachite green on Ichthyophthirius multifiliis Fouquet, 1876 (Hymenostomatida, Ciliophora) in ornamental fish.

For systemic therapy against trophozoites of the skin-inhabiting stage of Ichthyophthirius multifiliis in ornamental fish, the latter were fed medicated food flakes containing malachite green once daily for 1-11 days ad libitum. Naturally or artificially infected cardinal tetras (Paracheirodon axelrodi), blue gouramis (Trichogaster trichopterus), or clown loach (Botia macracantha) were used in the trials. The fish were maintained in aerated 12.5- or 60-1 aquaria at 23 degrees C. Ultrastructural investigations (scanning and transmission electron microscopy) revealed clear deleterious effects of malachite green on the parasitic stages. Following the initial application, the inner membrane of the mitochondria was destroyed. In fish fed for 2 days, aggregation of the mucocysts and polymerization the microtubules within the macronucleus occurred. Finally, the trophozoite's membrane was completely destroyed. In fish fed for 4 days, the medicated food killed all trophozoites of I. multifiliis. Sensitive ornamental fish (e.g., P. axelrodi) showed no adverse effects after they had been fed with only the medicated food flakes for 2 months. Therefore, the oral administration of malachite green using this newly developed medicated food considerably reduces the risk of toxic effects on the fish hosts, which are sometimes caused by malachite green following its application by immersion therapy. The feeding of flakes medicated with malachite green provides and easy-to-handle and highly effective treatment of I. multifiliis in ornamental fish.

Animal Feed↗
PubMed1976

Macronuclear response of Paramaecium multimicronucleatum to L-lysine.

A strain of Paramaecium multimicronucleatum was exposed to a medium containing L-lysine; the concentrations of the amino acid were 0.1%, 0.5% and 1.0% for different sets of experiments. In these two latter concentrations, the macronucleus of the ciliate broke down into innumerable small fragments, the microspheres. The micro-nuclei remained inert. The microspheres left the body of paramaecium as cell-free, self-duplicating entities constituted of DNA and RNA and enveloped by a protein coat. They had no nuclear membrane and they resembled the prokaryotes. Grown in culture medium with 0.1% horse serum, the microspheres transformed into small amoebae having typical eukaryotic features. These amoebae maintained a typical cyst-trophic cycle during the successive sub-cultures; they had no similarity with the paramaecia.

Animals↗
PubMed2005

Differential gene expression during stationary phase between amicronucleates and micronucleates of the ciliated protist, Pseudourostyla cristata.

Ciliates are unicellular eukaryotic organisms with two types of nuclei, the 'germline' micronucleus (MIC) and the 'somatic' macronucleus (MAC). We previously reported that when the MIC of Pseudourostyla cristata was eliminated by amputation, the resultant amicronucleate organisms exhibited a lower viability and abnormal oral structures. To gain insight into the genetic reorganization involved in or induced by removal of the MIC and the mechanism by which nuclear dimorphism was established, we investigated gene expression differences between amicronucleates and micronucleates, using suppression subtractive hybridization (SSH) techniques. Approximately 250 clones from each library were screened by cDNA array dot blotting. Altogether, 22 unique genes from the forward-subtractive library (micronucleates as tester, amicronucleates as driver) and 23 unique genes from the reverse-subtractive library (micronucleates as driver and amicronucleates as tester) were shown to be differentially expressed. These 45 differentially expressed genes were found to be homologs of genes involved in various cellular processes including signal transduction, transcription, cell cycle accomplishment and general metabolism, cell structure, and stress response. We highlighted 14 genes, 7 that were unique from both the forward-subtractive and the reverse-subtractive libraries, using real time semi-quantitative RT-PCR. The characterization of these cDNAs represents a starting point in understanding the molecular mechanisms of amicronucleates disruption.

Animals↗