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PubMed1984

DNA elimination in Tetrahymena: a developmental process involving extensive breakage and rejoining of DNA at defined sites.

Elimination of specific DNA sequences occurs during macronuclear development in the ciliate Tetrahymena thermophila. Recombinant DNA clones containing a segment of micronuclear (germinal) DNA involved in elimination and the corresponding segment of macronuclear (somatic) DNA produced after elimination were isolated. Detailed comparisons of the cloned DNAs, as well as the genomic DNAs, by hybridization indicated that DNA elimination is accompanied by specific DNA rearrangements. In this 9.5 kb region three defined DNA segments are deleted and the remaining sequences are linked together as one contiguous piece in the macronucleus. Specific DNA rearrangement of this kind occurs widely in the genome. Analysis of 20 randomly selected DNA clones suggests that there are more than 5000 such rearrangement sites in the genome. Thus specific breakage and rejoining of DNA occurs extensively during development, and might play an essential role in nuclear differentiation.

Animals↗
PubMed1984

Developmental rearrangements associated with a single type of expressed alpha-tubulin gene in Tetrahymena.

Southern blotting analyses with diverse heterologous probes indicate that there is only a single type of alpha-tubulin gene in the ciliated protozoan Tetrahymena thermophila. Comparisons of the germ-line configuration of alpha-tubulin sequences in the transcriptionally inert micronucleus with the somatic configuration in the transcriptionally active macronucleus indicate that rearrangements involving breakage and rejoining of sequences flanking both sides of the alpha-tubulin gene accompany macronuclear differentiation.

Animals↗
PubMed1985

The internally located telomeric sequences in the germ-line chromosomes of Tetrahymena are at the ends of transposon-like elements.

The germ-line micronuclear genome of the ciliate Tetrahymena thermophila contains approximately 10(2) chromosome-internal blocks of tandemly repeated C4A2 sequences (mic C4A2). This repeated sequence is the telomeric sequence in the somatic macronucleus. Each of six cloned micC4A2 was found to be adjacent to a conserved 30 bp sequence, which we propose is the terminal inverted repeat of a family of DNA elements (the Tel-1 family). This 30 bp sequence contains a site for the infrequently cutting restriction enzyme Bst XI, which allows full-length Tel-1 elements to be cut out of the micronuclear genome. BAL 31 exonuclease digestion of Bst XI-cut micronuclear DNA showed the majority of micC4A2 blocks to be associated with the ends of the Tel-1 family. We propose that Tel-1 elements are transposable and suggest a novel mechanism to account for the origin of micC4A2, in which telomeric repeats are added to the ends of free linear forms of the transposable elements prior to reintegration.

Animals↗
PubMed1985

Mobile elements bounded by C4A4 telomeric repeats in Oxytricha fallax.

A novel family of micronuclear elements termed telomere-bearing elements (TBEs) is described. All 1900 family members are eliminated during macronuclear development. We conclude that they are transposons, first because the members are moderately conserved in sequence and probably dispersed in the genome. Second, in two cases, sequence comparison of the termini and flanks of the element with the corresponding empty site indicate that elements cause 3 bp target duplications (AAT) upon insertion; the 3 bp are part of the 5 bp target sequence, AATGA. Lastly, both elements carry 77 or 78 bp inverted terminal repeats. The tip of each inverted terminal repeat is the 17 bp telomere-like sequence 5' C1A4C4A4C4. At least half of the elements have these 17 bp or an extremely similar sequence. One possible pathway for transposition into new micronuclear sites starts in the developing macronucleus with excision to create a free linear form to which telomeres are added, followed by a low frequency of movement to the micronucleus, and insertion into the germ-line micronuclear DNA.

Animals↗
PubMed1986

Control of rDNA replication in Tetrahymena involves a cis-acting upstream repeat of a promoter element.

A novel genetic scheme was used to isolate mutants altered in the formation or maintenance of amplified rDNA in the Tetrahymena macronucleus. One such mutant had a cis-acting rDNA mutation that affected the ability of mutant rDNA molecules to replicate in macronuclei in the presence of a wild-type (B strain) rDNA. The mutant rDNA was lost from these heterozygous macronuclei during vegetative cell divisions, although it was maintained normally in the homozygous or hemizygous state. In contrast, wild-type macronuclear rDNA of the C3 strain used to obtain the mutant outreplicated B strain rDNA in B/C3 heterozygote macronuclei. Sequence differences were found between wild-type B and C3 and mutant C3 rDNAs in the replication origin region, changing an upstream repeat of a highly conserved rRNA promoter element. We propose that the various rDNA alleles differentially compete for limiting amounts of trans-acting factors that bind to these enhancer-like repeats and positively regulate rDNA replication.

Animals↗
PubMed1987

A conserved nucleotide sequence at the sites of developmentally regulated chromosomal breakage in Tetrahymena.

Chromosomal breakage occurs at hundreds of specific sites in Tetrahymena, including the two ends of the unique ribosomal RNA genes, during the development of the somatic macronucleus. We have identified a 15-nucleotide sequence that occurs widely in the germinal micronuclear genome and is associated exclusively with chromosomal breakage sites. When copies of this sequence were cloned and analyzed, they were found in all cases to be located at or very near sites of breakage. This general rule is further supported by the observation that in a different site in which a single nucleotide substitution is found within this sequence, no chromosomal breakage occurs. The complete sequence structure of one of the breakage junctions has also been determined in both the germinal DNA and the two somatic DNA termini. This structure reveals that the 15-nucleotide conserved sequence is located within a 54-nucleotide region that is removed following chromosomal breakage.

Animals↗
PubMed1989

Developmentally coordinated en masse excision of a highly repetitive element in E. crassus.

The E. crassus Tec1 element is present in greater than 10(4) copies in the micronuclear genome but is absent from the macronuclear genome. During formation of a macronucleus from a micronucleus, a majority of the Tec1 elements appear as extrachromosomal circles. The circular and integrated forms of Tec1 have been characterized by restriction mapping to produce consensus maps and by sequence analysis of the element's termini. The circular forms are resistant to BAL31 and have the restriction map expected if the element excises at the end of its inverted repeats. DNA sequence analysis of a circular form confirms that the inverted repeats are in a head-to-head configuration. Excision of Tec1 occurs very early during macronuclear development as the DNA begins to replicate to form polytene chromosomes.

Animals↗
PubMed1989

Detection of circular forms of eliminated DNA during macronuclear development in E. crassus.

Following their sexual cycle, hypotrichous ciliated protozoa transform a copy of a chromosomal micronucleus into a macronucleus containing small, linear DNA molecules. A frequent event during macronuclear development is the removal of short segments of DNA (internal eliminated sequences: IESs) by a process equivalent to DNA breakage and rejoining. In this study we used a polymerase chain reaction procedure to demonstrate that free circular forms of IESs are present in cells undergoing macronuclear development. Sequencing of the junctions of the free circular IESs suggests that they share 12 nucleotides with the macronuclear DNA molecules that are generated following IES removal. The results provide evidence that IESs are removed by an active DNA breakage and rejoining process, which may involve staggered cuts in the substrate DNA.

Animals↗
PubMed1996

Developmentally programmed DNA rearrangement in Tetrahymena thermophila: isolation and sequence characterization of three new alternative deletion systems.

Extensive developmentally programmed DNA rearrangements, including thousands of internal deletions, occur in the differentiating somatic macronucleus in Tetrahymena thermophila. Some deletion systems involve the use of multiple alternative deletion sites. We report here the cloning and the sequences of three new alternative deletion systems (RR, RP and B) obtained using genomic subtraction. The RP and RR deletion systems are 2 kb apart on chromosome 1R, and both involve the removal of < 2 kb of micronuclear sequences. The B deletion system is on chromosome 5 and involves a deletion of > 5 kb. All three deleted regions are very AT rich (approximately 80%) and do not appear to encode any protein. Sequences of the regions flanking the deletion junctions of all three systems revealed no sequence similarity among them nor with any previously reported deletion systems, suggesting that different cis-acting elements are involved for rearrangement. Unlike other deletion systems in ciliates, the B deletion system lacks short terminal direct repeats. Our results suggest an average of at least one alternative deletion system per 134 kb of micronuclear DNA and lead to an estimate that at least 25% of all deletion systems in Tetrahymena utilize alternative ends. The genomic subtraction method employed in this study could prove useful for the isolation of alternatively deleted DNA in special-purpose cases in Tetrahymena and other ciliates. The hybridization parameters for genomic subtraction worked out here for highly AT-rich DNA may have wider usefulness.

Animals↗
PubMed1975

Nuclear characteristics and phylogeny in the protistan phylum Ciliophora.

Ciliates possess a number of nuclear characteristics which, in combination, are unique among the Protista. Nevertheless, attempts to understand the origin - presumably from a flagellate ancestry - of the Ciliophora as a phylum must be made, as well as efforts to elucidate phylogenetic pathways within the large and diverse assemblage represented by its present-day forms. The macronucleus may provide an important clue to early ciliate phylogeny, since we still have, among extant species, groups of distinct "karyological relicts" exhibiting the very features expected in hypothetical forms corresponding to postulated stages in macronuclear origin and evolution. The relationship of the groups of "relict" species to the predominant polyploid-macronucleate forms, with a direct impact on the classification system as well as ciliate evolution and phylogeny in general, is discussed in some detail. Arguments are presented for taxonomic separation of the relatively primitive homokaryotic and diploid-macronucleate forms, which also share other features in common related to their being members of the interstitial fauna, from the more advanced ciliates. The problem is complicated by the non-nuclear structural complexities of these sand-dwelling forms, apparently secondarily-derived specializations which, by convergence, have come to resemble certain non-homologous features of the allegedly more highly evolved groups.

Biological Evolution↗
PubMed1991

On the evolution of the karyorelict ciliate life cycle: heterophasic ciliates and the origin of ciliate binary fission.

Karyorelict ciliates have near diploid somatic nuclei (macronuclei) incapable of division. If selective pressure favors nuclear division, how could such macronuclei have evolved? I propose that they initially evolved in the context of a diplophase stage that consisted entirely of a non-dividing trophont that was terminated by the induction of meiosis. The diploid macronucleus then differentiated, functioned and was destroyed in the absence of cell division. Such a life cycle would necessarily be heterophasic, i.e. with alternating haploid and diploid generations. I call these ancestors heterophasic ciliates. I further propose that the ability of this diploid trophont to undergo binary fission arose de novo. Ciliate binary fission would then be a derived characteristic, which possibly evolved indepedently in more than one heterophasic ciliate lineage. A progression of steps, leading to the reduction of the haplophase and the generation of the karyorelict life cycle, is proposed. The shared possession of nuclear dimorphism with non-dividing macronuclei, conjugation, and a putative heterophasic ancestry invites further investigation of the phylogenetic relationship between heterokaryotic foraminifera and karyorelict ciliates.

Animals↗
PubMed1977

Effect of puromycin on synthesis, processing, and nucleocytoplasmic translocation of rRNA in Tetrahymena pyriformis.

1. Treatment of Tetrahymena pyriformis with various concentrations of puromycin results in a more pronounced inhibition of [3H]uridine accumulation in stable RNA than of protein synthesis. 2. At a concentration of 500 micrograms/ml, which is almost completely inhibitory to [3H]uridine incorporation in vivo, puromycin has no influence on the incorporation of [3H]UTP into RNA in isolated macronuclei. Pretreatment of the cells with the antibiotic, however, reduces the activity of RNA polymerases in isolated nuclei to less than 30%. 3. In puromycin-treated cells a small amount of pre-rRNA is synthesized but not processed into cytoplasmic rRNAs. 4. Puromycin reduces the nucleocytoplasmic translocation of pre-existing RNA to about 25% of the control rate within 5 min, resulting in an accumulation of relatively stable rRNA precursor molecules in the macronucleus.

Animals↗
PubMed1980

Inverted terminal repeat sequence in the macronuclear DNA of Stylonychia pustulata.

The structure of macronuclear DNA of a hypotrichous ciliate, Stylonychia pustulata, was examined by both electron microscopy and nucleotide sequence analysis. The DNA in the macronucleus consists of small linear molecules with average length of about 3400 base pairs (bp). Most, if not all, of these DNA molecules have identical inverted terminal repeat sequence of 20 nucleotide residues. This sequence is 5'-CCCCAAAACCCC-AAAACCCC.

Animals↗
PubMed1981

Extrachromosomal rDNA of Tetrahymena thermophila is not a perfect palindrome.

We have determined the restriction-endonuclease-cleavage map and the nucleotide sequence of the central 1.4 kb fragment of the macronuclear extrachromosomal rDNA of Tetrahymena thermophila. These data demonstrate that this molecule is not a perfect palindrome, having a 29 bp AT-rich non-palindromic sequence at its center. This observation is important in determining the mechanism by which a single chromosomally integrated rRNA gene in the micronucleus is rearranged and amplified during sexual development to yield multiple copies of extrachromosomal rDNA in the macronucleus.

Animals↗
PubMed1988

Replacement of the macronuclear ribosomal RNA genes of a mutant Tetrahymena using electroporation.

The macronucleus of the ciliate Tetrahymena contains approx. 10(4) ribosomal RNA gene molecules (rDNA) in the form of linear, autonomously replicating palindromes. Previous studies have shown that macronuclear rDNA molecules derived from wild-type (wt) inbred strain C3 out-replicate those derived from wt inbred strain B, in macronuclei initially heterozygous for both, leading to the complete loss of the B rDNA. However, rmm-1, a cis-acting laboratory-induced mutation obtained previously by mutagenesis of inbred strain C3, causes the mutant rmm-1 rDNA to be completely out-replicated by B rDNA. These findings suggest the following hierarchy of replication potential: wt C3 greater than wt B greater than C3-rmm-1. We used electroporation to test whether cells containing only rmm-1 macronuclear rDNA are favorable recipients for transformation with either wt B or C3 donor rDNA molecules. The donor rDNA molecules carried the selectable marker Pmr (paromomycin resistance) located in the coding region of the 17S rRNA. Transformants were obtained, at a frequency greater than 1 in 10(5), by electroporation under a wide range of electrical discharge parameters. The fraction of cells surviving electroporation varied between 2 and greater than 95% in successful experiments. Replacement ('transplacement') of the recipient rDNA was observed, consistent with the prediction that B and C3 rDNA should out-replicate rmm-1 rDNA. These findings are also consistent with the previous conclusion that the differential replication determinants reside in the 5'-nontranscribed spacer of the rDNA.

Animals↗
PubMed1989

Actin, tubulin and H4 histone genes in three species of hypotrichous ciliated protozoa.

In hypotrichous ciliated protozoa, genes are transcribed in the macronucleus where the genome consists of 'gene-sized' linear DNA molecules. We have isolated clones of actin, tubulin and H4 histone macronuclear genes from Oxytricha nova, Stylonychia lemnae and Euplotes crassus in an effort to determine if they possess molecules of similar size for a given coding function, and also to determine the size range of non-coding DNA present on these molecules. Our results indicate that while the length of their non-coding DNA can vary slightly, both between different hypotrichs and within the gene family of a single organism, actin and tubulin macronuclear molecules are similarly sized. The sizes observed for these molecules support the hypothesis that each macronuclear molecule encodes a single gene. However, the H4 histone macronuclear molecules show a much wider size range and generally are much longer than necessary to encode the H4 histone. We therefore sequenced a 1700-bp H4 histone macronuclear molecule from O. nova to determine if it might possibly encode additional gene products. Sequence data reveals the presence of nine open reading frames (ORFs) greater than 100 bp in length; however, Northern hybridization analysis of the products of this DNA molecule reveals only a single transcript.

Actins↗
PubMed1991

Structure and evolution of the Tetrahymena thermophila gene encoding ribosomal protein L21.

The single-copy gene encoding ribosomal protein (r-protein) L21 in the macronucleus of the ciliate Tetrahymena thermophila was cloned and characterized. Sequencing of the L21 gene and a corresponding cDNA clone showed the gene to contain three introns, all located in the 3' half of the coding region. Primer extension and nuclease protection analyses revealed five transcription start points (tsp) 56-73 nucleotides upstream from the start codon. The uppermost tsp mapped to the first T in a sequence, TATAA, often found at tsp in T. thermophila. A comparison of amino acid sequences of r-proteins revealed that T. thermophila L21 belongs to a family of r-proteins that have been conserved in eubacteria, archaebacteria and eukaryotes. On the basis of structural and functional considerations, we suggest that the eukaryotic, like the prokaryotic, members of this protein family interact with the 5S RNA complex in ribosomes.

Amino Acid Sequence↗