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PubMed1999

Faithful expression of a heterologous gene carried on an artificial macronuclear chromosome in Euplotes crassus.

Macronuclear chromosomes of hypotrichous ciliates are gene-sized molecules carrying the coding sequence flanked by short non-translated regions and bounded by telomeres. We have constructed artificial chromosomes for investigation of transcription in the macronucleus of Euplotes crassus. The neo gene was put under the control of the 5"-non-translated region of the TBP gene of E.crassus. These molecules were introduced into the cell with the help of liposomes. The cells were transformed and survived high concentrations of geneticin. The artificial chromosomes were kept in the macro-nucleus for at least 50 days at a copy number of about 200 per macronucleus. Expression of the gene was shown by reverse transcription of the neo messenger. The transcription start was mapped and found to coincide with that found on the natural macronuclear chromosome encoding TBP in E.crassus.

5' Untranslated Regions↗
PubMed2000

A family of developmentally excised DNA elements in Tetrahymena is under selective pressure to maintain an open reading frame encoding an integrase-like protein.

Tlr1 is a member of a family of approximately 20-30 DNA elements that undergo developmentally regulated excision during formation of the macronucleus in the ciliated protozoan TETRAHYMENA: Analysis of sequence internal to the right boundary of Tlr1 revealed the presence of a 2 kb open reading frame (ORF) encoding a deduced protein with similarity to retrotransposon integrases. The ORFs of five unique clones were sequenced. The ORFs have 98% sequence conservation and align without frameshifts, although one has an additional trinucleotide at codon 561. Nucleotide changes among the five clones are highly non-random with respect to the position in the codon and 93% of the nucleotide changes among the five clones encode identical or similar amino acids, suggesting that the ORF has evolved under selective pressure to preserve a functional protein. Nineteen T/C transitions in T/CAA and T/CAG codons suggest selection has occurred in the context of the TETRAHYMENA: genome, where TAA and TAG encode Gln. Similarities between the ORF and those encoding retrotransposon integrases suggest that the Tlr family of elements may encode a polynucleotide transferase. Possible roles for the protein in transposition of the elements within the micronuclear genome and/or their developmentally regulated excision from the macronucleus are discussed.

Amino Acid Sequence↗
PubMed2002

A novel family of mobile genetic elements is limited to the germline genome in Tetrahymena thermophila.

In the ciliated protozoan Tetrahymena thermophila, extensive DNA elimination is associated with differentiation of the somatic macronucleus from the germline micronucleus. This study describes the isolation and complete characterization of Tlr elements, a family of approximately 30 micronuclear DNA sequences that are efficiently eliminated from the developing macronucleus. The data indicate that Tlr elements are comprised of an approximately 22 kb internal region flanked by complex and variable termini. The Tlr internal region is highly conserved among family members and contains 15 open reading frames, some of which resemble genes encoded by transposons and viruses. The Tlr termini appear to be long inverted repeats consisting of (i) a variable region containing multiple direct repeats which differ in number and sequence from element to element and (ii) a conserved terminal 47 bp sequence. Taken together, these results suggest that Tlr elements comprise a novel family of mobile genetic elements that are confined to the Tetrahymena germline genome. Possible mechanisms of developmentally programmed Tlr elimination are discussed.

Amino Acid Sequence↗
PubMed1987

Alternative processing during development of a macronuclear chromosome family in Oxytricha fallax.

Macronuclear chromosomes in Oxytricha fallax, a hypotrichous ciliate, are very short. They often belong to small families of cross-hybridizing chromosomes of two or three different sizes. For example, the 81-MAC family consists of three sizes of macronuclear chromosomes (4.9, 2.9, and 1.6 kbp) (Cartinhour and Herrick 1984). We show that the family actually consists of two closely related sets of three each and that the two sets are independently created by alternative processing of two separate precursor (micronuclear) versions. Chromosomes of a set share a common 1.6-kbp region, which contains a transcribed gene coding for a 25-kD protein. Different-sized macronuclear chromosomes of a set result from alternative choices of positions for telomere formation. All six members of the family are reproducibly generated in each developing macronucleus, and their copy numbers are stably maintained during vegetative replication of the macronucleus (Herrick et al. 1987). Here we argue for the existence of three distinct copy control elements in the 81-MAC family chromosomes. A model is discussed in which, following polytenization of the micronuclear chromosomes, different chromatids are processed differently, and, subsequently, replication-competent macronuclear chromosome products are amplified under the influence of the vegetative copy control elements.

Amino Acid Sequence↗
PubMed1992

Analysis of a scrambled gene: the gene encoding alpha-telomere-binding protein in Oxytricha nova.

Following cell mating in ciliates, a copy of the micronuclear genome is processed into a new macronucleus through massive cutting, reordering, splicing, elimination, and amplification of the DNA. DNA processing includes the deletion of short interrupting elements called internal eliminated sequences (IESs), followed by the splicing of the remaining segments, known as macronuclear destined sequences (MDSs). The MDSs in some micronuclear genes, such as actin I, are scrambled and must be reordered during IES removal and MDS splicing to yield a functional gene. Here, we describe the cloning, sequencing, and characterization of a different scrambling pattern for the gene that encodes the alpha subunit of the telomere-binding protein of Oxytricha nova. The micronuclear gene is made up of 14 MDSs in the scrambled order 1-3-5-7-9-11-2-4-6-8-10-12-13-14. Only the scrambled version is present in the micronucleus, and only the unscrambled version is present in the macronucleus. We propose that unscrambling occurs by homologous recombination guided by pairs of direct repeats at MDS-IES junctions. The patterned array of scrambling may be a clue to the origin of scrambling in this gene.

Animals↗
PubMed1995

An unusual sequence arrangement in the telomeres of the germ-line micronucleus in Tetrahymena thermophila.

The ciliated protozoan Tetrahymena thermophila contains two nuclei that differ dramatically in function, chromosome size and number, chromatin structure, and mode of division. It is possible that the telomeres of the two nuclei have different functions. Although macronuclear telomeric DNA has been well characterized and consists of tandem G4T2/C4A2 repeats that are synthesized by the enzyme telomerase, micronuclear telomeres have not been isolated previously. Here, we report the identification and cloning of micronuclear telomeres and demonstrate that although they contain the same terminal tandem G4T2 repeats as macronuclear telomeres, they are strikingly different in three respects. First, the tracts of G/C-rich telomeric repeats are approximately seven times longer in the micronucleus than in the macronucleus (approximately 2.0-3.4 vs. approximately 0.3-0.5 kb, respectively) from the same cell population. Second, the immediate telomere-associated sequences (TASs) from six different micronuclear chromosome ends have an unusually high G/C content and degree of homology to one another, unlike macronuclear TASs. The TAS from at least one micronuclear chromosome is unique to micronuclear telomeres and is not present in the macronucleus. Finally, and unexpectedly, all micronuclear telomere clones contain an inner homogeneous tract of a variant G4T3 repeat adjacent to the distal tract of G4T2 repeats. The native micronuclear telomeric DNA is composed of approximately 30% G4T3 repeats, corresponding to 0.6-1.0 kb per average telomere, positioned centromere-proximally to most or all of the G4T2 repeats. Neither the G4T3 sequence nor any other variant repeat is found in macronuclear telomeres. Furthermore, such a homogeneous tract of a variant repeat has not been found in the telomeres of any eukaryote.

Animals↗
PubMed1987

Multiple sequence versions of the Oxytricha fallax 81-MAC alternate processing family.

The 81-MAC family consists of three sizes of macronuclear chromosomes in Oxytricha fallax. Clones of these and of micronuclear homologs have been classified according to DNA sequence into three highly homologous (95.9-97.9%), but distinct versions. Version A is represented by a micronuclear clone and by clones of two different-sized macronuclear chromosomes, showing that alternate processing of micronuclear DNA is responsible for the variety of sizes of macronuclear chromosomes. Three Internal Eliminated Sequences (IES's) are demonstrated in Version A micronuclear DNA. Two have been sequenced and show short, flanking direct repeats but no inverted terminal repeats. Version C micronuclear DNA has interruptions in the macronuclear homology which correspond closely to the Version A IES's. Whether they are true IES's is unknown because no Version C macronuclear DNA has been demonstrated. Version C micronuclear DNA may be "macronuclear-homologous" but "micronucleus-limited" and not "macronucleus-destined." Version B is represented by macronuclear DNA clones, but no micronuclear clones. Vegetative micronuclear aneuploidy is suggested. The possible role of micronuclear defects in somatic karyonidal senescence is discussed in light of the precise macronuclear chromosome copy controls demonstrated within the 81-MAC family. These controls apparently operate throughout karyonidal life to maintain 1) a constant absolute amount of 81-MAC sequences in the macronucleus and 2) a constant stoichiometry within the family, both according to version and chromosome size.

Animals↗
PubMedDate not supplied

Non-coding DNA in macronuclear chromosomes of hypotrichous ciliates.

Massive elimination of sequences occurs in the development of the macronucleus of hypotrichous ciliates. The surviving sequences are presumed to have functions in the macronucleus; what little is known about non-coding macronuclear sequences is reviewed. The 1.7 kbp macronuclear chromosome that carries a histone H4 gene consists primarily of non-coding DNA 5' of the histone gene. This region is shown by sequence comparison to carry several perfectly conserved sequence blocks up to 14 bp long, scattered amongst regions which have evolved greatly since the divergence of Oxytricha nova and Stylonychia lemnae. This result is consistent with the suggestion of Harper and Jahn [Harper, D. S. & Jahn, C. L. 1989. Actin, tubulin and H4 histone genes in three species of hypotrichous ciliated protozoa. Gene, 75:93-107] that this large non-coding 5' region may be involved in the transcriptional regulation of the histone H4 gene carried on the 1.7 kbp chromosome. Very little is known about transcriptional control in hypotrichs; identification of conserved non-coding sequences of orthologous hypotrich genes promises to provide clues to potential cis-acting control signals.

Animals↗
PubMedDate not supplied

A method for the amplification of Paramecium micronuclear DNA by polymerase chain reaction and its application to the central repeats of Paramecium primaurelia G surface antigen genes.

This paper describes a method which allows the amplification of Paramecium micronuclear DNA. Amacronucleate cells are first obtained by an appropriate treatment with nocodazole, a microtubule depolymerizing agent which blocks the elongation of the macronucleus and the distribution of the micronuclei at cell division between the two daughter cells; then, DNA from such cells is amplified by the polymerase chain reaction technique. We have applied this method to the problem of the central repeats of the G surface antigen of P. primaurelia (strain 156). The central repeats consist of a 74 amino acid sequence repeated in tandem. The sequence identity of these repeats is also found in the nucleotide sequence even at silent codon positions, suggesting the existence of a mechanism of identity maintenance acting at the nucleotide level. Mechanisms based on RNA secondary structure which are frequently proposed as an explanation of this phenomenon are unlikely to be valid in this case. One can, therefore, imagine that these repeats might originate from one micronuclear sequence through duplicative processes which could occur during the formation of the macronucleus. We have used the described technique to amplify the micronuclear version of the central repeats and showed that it is identical to the macronuclear version, thus ruling out the above hypothesis. Therefore, intragenic recombination appears to be the most likely explanation of the sequence identity of these central repeats.

Amino Acid Sequence↗
PubMedDate not supplied

Induction of antibiotic resistance in Paramecium tetraurelia by the bacterial gene APH-3'-II.

We have generated a transformation marker for Paramecium using a Paramecium expression vector (pPXV) and the open reading frame (ORF) of the bacterial antibiotic resistance gene aminoglycoside 3'-phosphotransferase-II (APH-3'-II or neor) from the transposon Tn5. The expression vector contained a small multiple cloning site between the 5' and 3' non-coding regions of the calmodulin gene, and Tetrahymena telomere sequences for the stability of the plasmid in Paramecium. After the neor ORF was inserted, the plasmid was referred to as pPXV-NEO. Delivery of approximately 10-20 picoliters of linearized PXV-NEO at > or = 2000 copies/pl into the macronucleus effected 100% transformation. Southern and Northern blot hybridization showed the presence of neor-specific DNA and RNA, respectively, in all of the transformed clones but not in the untransformed clones. The degree of resistance to G-418, and the concentrations of neor-specific DNA and neor-specific RNA in the clones were proportional to the concentration of the vector injected. We have demonstrated that when the linearized plasmid was injected into the macronucleus, the prokaryotic sequence conferred an antibiotic resistance to Paramecium despite codon-usage differences.

Animals↗
PubMedDate not supplied

Invention and mystery in hypotrich DNA.

Hypotrichs have evolved extraordinary ways of organizing, manipulating, and replicating the DNA in their micronuclear and macronuclear genomes. Short macronuclear DNA molecules containing single genes are created by excision from chromosomes, accompanied by massive elimination of the germline DNA sequences between genes. Germline genes themselves are interrupted by multiple noncoding segments called internal eliminated segments, or IESs, that divide genes into multiple macronuclear-destined segments, or MDSs. The functional significance of this organization is unknown. Over evolutionary time IESs accumulate mutations rapidly, are inserted into or excised from genes, and shift position along DNA molecules. MDSs are ligated to create functional genes when IESs are spliced out of micronuclear DNA during macronuclear development. MDSs in some germline genes are in scrambled disorder and become unscrambled in association with IES elimination. Replication of DNA in the macronucleus is accomplished by organization of replication enzymes and factors into a structure that sweeps through the macronucleus to replicate the many millions of gene-sized DNA molecules. The significance of many of the bizarre DNA phenomena in the evolutionary/functional success of hypotrichs is still unclear.

Animals↗
PubMedDate not supplied

Nuclear death in living Tetrahymena: the case of the haploid nuclei.

During sexual conjugation in Tetrahymena the micronucleus divides meiotically, producing four haploid nuclei. While one of these nuclei divides mitotically to yield two genetically identical gametic pronuclei, a stationary pronucleus and a migratory pronucleus, the remaining three haploid nuclei degenerate and disappear. Typically, they migrate to the posterior end of the cell where they remain as residual bodies until they disappear. In the present study we asked whether degenerating haploid nuclei share any properties with apoptotic nuclei. Specifically, we wondered whether they would be stained by "apofluor", a combination of vital fluorescent indicators that differentially stains apoptotic nuclei in living cells. "Apofluor" includes acridine orange, which becomes trapped in acidic compartments and stains lysosomal bodies a brilliant orange-red, and Hoechst 33342, which binds to DNA and stains nuclei bright blue. With this dye combination, while ordinary nuclei stain blue, the apoptotic macronucleus stains first blue-green, then yellow, and finally orange. The progression in color is presumed to be due to the accumulation of protons in the apoptotic nucleus compartment. We found that three of the four post-meiotic haploid nuclei, those that are eliminated, were stained differentially green, then yellow, and then come to be indistinguishable from the orange lysosomal bodies. Differential staining can occur even while the nuclei are located at the anterior ends of the cells, and before the "viable" nucleus divides to form pronuclei. These results indicate that haploid nuclei in the process of degradation are differentially stained in living cells by "apofluor", and that the differential staining occurs early in the elimination process. Further, since the degenerating haploid nuclei are stained by "apofluor" it is likely that they are degraded by a mechanism similar to the elimination of the apoptotic macronucleus.

Animals↗
PubMedDate not supplied

Profilin functions in cytokinesis, nuclear positioning, and stomatogenesis in Tetrahymena thermophila.

Expression of the actin-binding protein profilin was disrupted in the ciliate Tetrahymena thermophila by an antisense ribosome method. In cells with the antisense disruption no profilin protein was detected. Cultures of cells with the antisense disruption could be maintained, indicating that profilin was not essential for cytokinesis or vegetative growth. Disruption of the expression of profilin resulted in many cells that were large and abnormally shaped. Formation of multiple micronuclei, which divide mitotically, was observed in cells with a single macronucleus, indicating a defect in early cytokinesis. Some cells with the antisense disruption contained multiple macronuclei, which in Tetrahymena may indicate a function late in cytokinesis. The lack of profilin also affected cytokinesis in the cells that could divide. Normal-sized and normal-shaped cells with the antisense disruption took significantly longer to divide than control cell types. The profilin disruption revealed two new processes in which profilin functions. In cells lacking profilin, micronuclei were not positioned at their normal site on the surface of the macronucleus and phagocytosis was defective. The defect in phagocytosis appeared to be due to disruption of the formation of oral apparatuses (stomatogenesis) and a possible failure in the internalization of phagocytic vacuoles.

Actins↗
PubMedDate not supplied

Redescription of Lagenophrys cochinensis Santhakumari & Gopalan, 1980 (Ciliophora, Peritrichia, Lagenophryidae), an Ectosymbiont of Marine Isopods, including new information on morphology, geographic distribution, and intraspecific variation.

Lagenophrys cochinensis is a peritrich ciliate originally reported as an ectocommensal of the wood-boring isopods Sphaeroma terebrans, Sphaeroma triste, and Sphaeroma annandalei and the tanaidacean Apseudes chilkensis in estuaries of southern India. In the present study, it was found to occur also on Sphaeroma quoyanum, Sphaeroma walkeri, and Exosphaeroma planulum. New material was used to make permanent preparations, allowing a comprehensive description of the morphology of L. cochinensis for the first time. The macronucleus of L. cochinensis was found to have an elongate shape that spans the width of the cell body, unlike the compact macronucleus originally described. In addition, the loricae of all samples examined were subcircular or shorter than wide, not longer than wide as originally described. Polykinetid 3 of the infundibular infraciliature consisted of three rows of kinetosomes, only the third species of Lagenophrys found to have more than two rows in polykinetid 3 so far. Samples of L. cochinensis on S. quoyanum from New Zealand and California appeared to represent a population distinct from others. The species has a cosmopolitan distribution, probably owing to the ease with which its hosts are transported from one estuary to another in drifting wood or on hulls of ships.

Animals↗
PubMedDate not supplied

Two new species of Epistylis (Ciliophora: Peritrichida) on the blue crab (Callinectes sapidus) in the Gulf of Mexico.

Two epibiotic peritrichs infested the blue crab, Callinectes sapidus, from the Gulf of Mexico, Mississippi, USA. Epistylis callinectes n. sp. was isolated from the epipods of maxillipeds, bases of gill-cleaning setae, and gills, and Epistylis clampi n. sp. was isolated from the exterior surfaces of the exoskeleton. Epistylis callinectes has short, symmetrically and dichotomously branched stalks; its zooid is elongate ovoid and conspicuously longer than the individual stalk branches, measuring 40-57 (49) x 18-33 (26) microm in vivo and containing a thick, undivided peristomial lip (PL). It has a single contractile vacuole and a transverse horseshoe-shaped macronucleus. Its haplokinety (H) and polykinety (Po) complete one and one-half circuits on the peristome before entering the infundibulum. There is a distal kinetal fragment present at the distal end of both the H and Po. Epistylis callinectes has 48-70 transverse silverlines from the oral area to the trochal band (TB) and 19-26 from the TB to the scopula. Epistylis clampi has long, asymmetrically, and dichotomously branched stalks. Its zooid is elongate vase-shaped, measuring 35-64 (48) x 21-30 (27) microm in vivo and with a thick, transversely folded PL. The stalks supporting zooids are unequal in length. Its zooid has a single contractile vacuole and a transverse horseshoe-shaped macronucleus occurs in the upper half of the body. Its H and Po complete approximately one circuit around the peristome before entering the infundibulum. There is a distal kinetal fragment present at the distal end of both the H and Po. This species has 71-112 transverse silverlines from the peristome to the scopula.

Animals↗
PubMed1993

Effects of immobilization on growth, morphology, and DNA content of the ciliated protozoon Tetrahymena thermophila.

Morphological and physiological properties of Tetrahymena thermophila immobilized by encapsulation in calcium-alginate hollow spheres were found to be substantially different from those of suspended cells. Immobilized T. thermophila reached lengths of 70-100 microns, whereas the average cell of suspension cultures was about 40 microns long. Suspended cells appeared typically pear-shaped while immobilized cells developed a proboscis-like anterior end. Contrary to suspended T. thermophila, encapsulated cells were functionally deficient in phagocytosis although developing an oral apparatus. The diameter of the macronucleus of immobilized cells was about two times larger than the macronucleus of suspended cells and contained twice as much DNA, while the DNA content of the micronucleus remained unchanged. High cell density fermentations of suspended cells indicated that the alterations observed in immobilized cells were not due to close physical contacts between the cells.

Animals↗
PubMed1999

Effects of antibiotics on the early infection process of a macronuclear endosymbiotic bacterium Holospora obtusa of Paramecium caudatum.

We examined the effects of antibiotics involved in bacterial DNA, RNA and protein synthesis and host protein synthesis on the early infection process of the bacterium Holospora obtusa, a macronucleus-specific symbiont of the ciliate Paramecium caudatum. Infection of the host macronucleus by the bacterium was not inhibited by mitomycin C, rifampicin and chloramphenicol. However, ingestion of the bacterium into the host digestive vacuoles and escape of the bacterium from the vacuoles to the host cytoplasm were significantly arrested with emetine. The results suggest that newly synthesized host proteins play an important role in the early infection process.

Animals↗
PubMed1999

Evolution of DNA organization in hypotrichous ciliates.

Profound changes have been introduced into the germline (micronuclear) and somatic (macronuclear) genomes of hypotrichous ciliates during evolution. First, multiple, short, unique, noncoding sequences, called IESs, have been inserted into micronuclear genes. IESs are spliced out of each gene, and the gene segments, called MDSs, are ligated during conversion of the micronuclear genome to a macronuclear genome after cell mating. The IESs in a particular gene can change dramatically in number, position, length, and sequence during speciation. Once inserted, IESs can shift along the DNA of a gene 1 or 2 bp at a time by a mutational mechanism that does not alter the coding sequence of the gene. Second, the MDSs in the same genes have been rearranged into random or nonrandom, scrambled disorder. The origin of nonrandom scrambling patterns can be explained by a model of simultaneous insertion of multiple IESs into a germline gene during evolution. Subsequent recombinations among the IESs may change the MDS arrangement from a nonrandomly scrambled to a randomly scrambled pattern, including inversions of MDSs. Third, during the conversion of a micronucleus to a macronucleus after cell mating, MDSs are ligated in the unscrambled, orthodox order in association with IES excision, and the genes are then removed from the chromosomes as individual, short DNA molecules. These gene-size molecules are amplified many-fold to produce a mature macronucleus. All of these phenomena attest to a remarkable fluidity of the hypotrich genome both over evolutionary time and in the conversion of a germline genome to a somatic genome. The significance of this fluidity for the life and evolution of these organisms is still obscure. Recombination among IESs could shuffle MDSs and facilitate faster evolution of new genes.

Animals↗