Germ Line-Specific DNA Sequences are Present on All Five Micronuclear Chromosomes in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

The development of the macronucleus from the zygotic micronucleus in the ciliated protozoan Tetrahymena spp. involves the elimination of specific DNA sequences (M. C. Yao and M. Gorovsky, Chromosoma 48:1-18 1974). The present study demonstrates that micronucleus-specific DNA is present on all five of the micronuclear chromosomes. Fragments of micronuclear DNA from Tetrahymena thermophila were cloned in the plasmid vector pBR322. A procedure was developed to examine the organization of the cloned sequences in micro- and macronuclear DNA without nick translating each individual probe. Twenty-three percent of randomly selected DNA sequences examined by this method were micronucleus (germ line) specific. They were all members of families of repeated sequences. Hybridization of six micronucleus-specific DNA sequences to micronuclear DNA from nullisomic strains of T. thermophila, which are lacking one or more pairs of chromosomes in the micronucleus, suggested that these sequences are present on several chromosomes. One micronucleus-specific sequence was shown by in situ hybridization to be present on all five of the micronuclear chromosomes

DNA Synthesis, Methylation and Degradation During Conjugation in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

We have investigated the timing of DNA synthesis, methylation and degradation during macronuclear development in the ciliate, Tetrahymena thermophila. DNA synthesis was first detected in the anlagen early in macronuclear development, but the majority of DNA synthesis occurred later, after pair separation. Anlagen DNA was first detectably methylated at GATC sites 3–5 hours after its synthesis. Once initiated, de novo methylation was rapid and complete, occurring between 13.5 and 15 hours of conjugation. The level of methylation of GATC sites was constant throughout the remainder of conjugation, and was similar to that in mock-conjugated cells. Degradation of DNA in the old macronucleus and DNA synthesis in the anlagen began at about the same time. Upon pair separation, less than 20% of old macronuclear DNA remained. A small percentage of nucleotides prelabeled prior to conjugation were recycled in the developing analgen

Diverse Sequences within Tlr Elements Target Programmed DNA Elimination in \u3cem\u3eTetrahymena Thermophila\u3c/em\u3e

Tlr elements are a novel family of ~30 putative mobile genetic elements that are confined to the germ line micronuclear genome in Tetrahymena thermophila. Thousands of diverse germ line-limited sequences, including the Tlr elements, are specifically eliminated from the differentiating somatic macronucleus. Macronucleusretained sequences flanking deleted regions are known to contain cis-acting signals that delineate elimination boundaries. It is unclear whether sequences within deleted DNA also play a regulatory role in the elimination process. In the current study, an in vivo DNA rearrangement assay was used to identify internal sequences required in cis for the elimination of Tlr elements. Multiple, nonoverlapping regions from the ~23-kb Tlr elements were independently sufficient to stimulate developmentally regulated DNA elimination when placed within the context of flanking sequences from the most thoroughly characterized family member, Tlr1. Replacement of element DNA with macronuclear or foreign DNA abolished elimination activity. Thus, diverse sequences dispersed throughout Tlr DNA contain cis-acting signals that target these elements for programmed elimination. Surprisingly, Tlr DNA was also efficiently deleted when Tlr1 flanking sequences were replaced with DNA from a region of the genome that is not normally associated with rearrangement, suggesting that specific flanking sequences are not required for the elimination of Tlr element DNA

A Family of DNA Sequences is Reproducibly Rearranged in the Somatic Nucleus of \u3cem\u3eTetrahymena\u3c/em\u3e

A small family of DNA sequences Is rearranged during the development of the somatic nucleus in Tetrahymena. The family is defined by 266 bp of highly conserved sequence which restriction mapping, hybridization and sequence analysis have shown is shared by a cloned micronuclear fragment and three sequences which constitute the macronuclear family. Genomic Southern hybridization experiments indicate there are five members of the family in micronuclear DNA. All of the family members are present in whole genome homozygotes and are therefore nonallellic. The three macronuclear sequences are all present in clonal cell lines and are reproducibly generated in every developing macronucleus. The rearrangement event begins 14 hours after conjugation is initiated and is nearly completed by 16 hours

Methylation of Replicating and Nonreplicating DNA in the Ciliate \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

Methylation of adenine in replicating and nonreplicating DNA of the ciliate Tetrahymena thermophila was examined. In growing cells, 87% of the methylation occurred on the newly replicated daughter strand, but methylation was also detectable on the parental strand. Methylation of nonreplicating DNA from starved cells was demonstrated

A Family of Developmentally Excised DNA Elements in \u3cem\u3eTetrahymena\u3c/em\u3e is under Selective Pressure to Maintain an Open Reading Frame Encoding an Integrase-Like Protein

Tlr1 is a member of a family of ~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 TIC transitions in T/CAA and T/CAG codons suggest selection has occurred in the context of the Tetrahymena genome, where TAA and TAG encode Gin. 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

A Developmentally Regulated Deletion Element with Long Terminal Repeats Has \u3cem\u3eCis\u3c/em\u3e-Acting Sequences in the Flanking DNA

Approximately 6000 specific DNA deletion events occur during development of the somatic macronucleus of the ciliate Tetrahymena. The eliminated Tlr1 element is 13 kb or more in length and has an 825 bp inverted repeat near the rearrangement junctions. A functional analysis of the cis-acting sequences required for Tlr1 rearrangement was performed. A construct consisting of the entire inverted repeat and several hundred base pairs of flanking DNA on each side was rearranged accurately in vivo and displayed junctional variability similar to the chromosomal Tlr1 rearrangement. Thus, 11 kb or more of internal element DNA is not required in cis for DNA rearrangement. A second construct with only 51 bp of Tetrahymena DNA flanking the right junction underwent aberrant rearrangement. Thus, a signal for determination of the Tlr1 junction is located in the flanking DNA, 51 bp or more from the right junction. Within the Tlr1 inverted repeat are 19 bp tandem repeats. A construct with the 19mer repeat region deleted from the right half of the inverted repeat utilized normal rearrangement junctions. Thus, despite its transposon-like structure, Tlr1 is similar to other DNA rearrangements in Tetrahymena in possessing cis-acting sequences outside the deleted DNA

Transformation of \u3cem\u3eTetrahymena thermophila\u3c/em\u3e with Hypermethylated rRNA Genes

The extrachromosomal rRNA genes (rDNA) of Tetrahymena thermophila contain 0.4% N6-methyladenine. C3 strain rDNA was isolated, hypermethylated in vitro, and microinjected into B strain host cells. Clonal cell lines were established, and transformants were selected on the basis of resistance to paromomycin, conferred by the injected rDNA. The effects of methylation by three enzymes which methylate the sequence 5\u27-NAT-3\u27, the dam, EcoRI, and ClaI methylases, were tested. Hypermethylation of the injected rDNA had no effect on transformation efficiency relative to mock-methylated controls. The injected C3 strain rDNA efficiently replaced host rDNA as the major constituent of the population of rDNA molecules. Hypermethylation of the injected DNA was not maintained through 20 to 25 cell generations

\u3cem\u3eASI1\u3c/em\u3e, a Gene Encoding a Novel Leucine Zipper Protein, Is Induced during Development of the Macronucleus in \u3cem\u3eTetrahymena\u3c/em\u3e

Sexual reproduction in the ciliate Tetrahymena follows a complex developmental program involving the sequential regulation of dozens of genes. Genes that are up-regulated during post-zygotic development in Tetrahymena were isolated by subtractive hybridization. Anlagen stage induced gene 1 (ASI1) encodes a 2.8 kb transcript that contains a single intron and is induced during macronuclear development. ASI1 is a single copy gene in both the micronucleus and the macronucleus. It encodes a 95 kDa conceptual protein with a leucine zipper near the amino terminu

Position Effect Takes Precedence Over Target Sequence in Determination of Adenine Methylation Patterns in the Nuclear Genome of a Eukaryote, \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

Approximately 0.8% of the adenine residues in the macronuclear DNA of the ciliated protozoan Tetrahymena thermophila are modified to N6-methyladenine. DNA methylation is site specific and the pattern of methylation is constant between clonal cell lines. In vivo, modification of adenine residues appears to occur exclusively in the sequence 5′-NAT-3′, but no consensus sequence for modified sites has been found. In this study, DNA fragments containing a site that is uniformly methylated on the 50 copies of the macronuclear chromosome were cloned into the extrachromosomal rDNA. In the novel location on the rDNA minichromosome, the site was unmethylated. The result was the same whether the sequences were introduced in a methylated or unmethylated state and regardless of the orientation of the sequence with respect to the origin of DNA replication. The data show that sequence is insufficient to account for site-specific methylation in Tetrahymena and argue that other factors determine the pattern of DNA methylation