DNA synthesis, methylation and degradation during conjugation in Tetrahymena thermophila

DNA in the polyploid macronucleus of the ciliated protozoan Tetrahymena thermophila contains the modified base N6-methyladenine. We identified two GATC sites which are methylated in most or all of the 45 copies of the macronuclear genome. One site is 2 kilobases 5' to the histone H4-I gene, and the other is 5 kilobases 3' to the 73-kilodalton heat shock protein gene. These sites are de novo methylated between 10 and 16 h after initiation of conjugation, during macronuclear anlage development. The methylation states of these two GATC sites and four other unmethylated GATC sites do not change in the DNA of cells cultured under conditions which change the activity of the genes, including logarithmic growth, starvation, and heat shock.The nuclear DNAs of most eucaryotes contain the modified base 5-methylcytosine (5MeC) as a minor component. In many cases, site-specific methylation of cytosine, particularly near the 5' ends of genes, has been correlated with gene inactivity (for reviews see references 1, 17, and 45). Therefore, it has been suggested that 5MeC may be involved in transcriptional regulation.N6-methyladenine (N6MeA) is found in the nuclear DNAs of several unicellular eucaryotes, either in combination with other modifications or as the only modified base. The DNAs of the green algae Chlamydomonas reinhardi and Chlorella spp. have both methylcytosine and methyladenine (28, 46), while only methylcytosine has been found in the DNA of another green alga, Euglena gracilis (40). In the ciliates Tetrahymena (23), Paramecium (16), Oxytricha (39), and Stylonychia (5) N6MeA is the sole modified base. The function of N6MeA in eucaryotic DNA is unknown.We examined methylation at specific sites in the genome of Tetrahymena thermophila. Tetrahymena cells contain two nuclei, a diploid micronucleus and a polyploid macronucleus. The macronucleus is responsible for most, if not all, transcriptional activity during vegetative growth. During sexual reproduction (conjugation), the macronucleus is destroyed, and mitotic products of the zygotic micronucleus develop into a new micronucleus and a new macronucleus. The developing macronucleus, called the anlage, undergoes many structural and morphological changes, including DNA replication to a final DNA content of 45c (49), elimination and rearrangement of germ line DNA sequences (50), and de novo methylation (9, 26).Although the micronuclear DNA of Tetrahymena cells does not contain modified bases, 0.8% of the adenine residues in the transcriptionally active macronucleus are modified to N6MeA (23,28). A partially purified methylase activity was isolated from macronuclei (10). A nearestneighbor analysis of in vivo methylated DNA showed that all four bases are found 5' to the methylated adenine, but only thymidine is found 3'. Some of the methyladenine residues in Tetrahymena macronuclear DNA occur in the sequence * Corresponding author. t Present address: Eleanor Roosevelt Cancer Institute, Denver, CO 80262.5'-GMeATC-3' and can be assayed by digestion with restriction enzyme DpnI...

mentioning

confidence: 99%

“…Methylation would then be expected to precede or correlate with the onset of transcription in the developing macronuclear anlagen. De novo methylation of bulk anlage DNA at GATC sites is first detectable between 13.5 and 15 h after initiation of conjugation, approximately midway through anlage development (9,26).…”

mentioning

confidence: 99%

Site-specific methylation of adenine in the nuclear genome of a eucaryote, Tetrahymena thermophila.

Harrison¹,

Findly²,

Karrer³

1986

“…In Tetrahymena thermophila, 0.6 to 0.8% of the adenines are methylated (5), methylation occurring at the sequence 5' AT 3' (3). Methyladenines are only present in the pseudopolyploid somatic macronucleus, which is transcriptionally active, and not in the diploid germinal micronucleus, which is transcriptionally silent (5,7). As a new macronucleus develops after conjugation, the chromosomes are fragmented and micronuclear DNA sequences are eliminated (reviewed in reference 4).…”

mentioning

confidence: 99%

“…As a new macronucleus develops after conjugation, the chromosomes are fragmented and micronuclear DNA sequences are eliminated (reviewed in reference 4). At the same time, DNA becomes methylated (7). Within the ribosomal DNA of the macronucleus, all identified GATC sites are methylated on both strands; however, any particular rDNA molecule is only methylated at one or a few of the possible methylation sites (2).…”

mentioning

confidence: 99%

Methylation site within a facultatively persistent sequence in the macronucleus of Tetrahymena thermophila.

White¹,

McLaren²,

Allen³

1986

“…The 21 cpm measured at the origin probably line previously corresponded to partially hydrolyzed DNA. Thus, label DNA (7). This incorporated into the DNA of starved cells was the result of of DNA from adenine methylation.…”

mentioning

confidence: 99%

Methylation of replicating and nonreplicating DNA in the ciliate Tetrahymena thermophila.

Harrison¹,

Karrer²

1989

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.The modified base N6-methyladenine (N6MeA) is present as a minor component in DNA from several unicellular eucaryotes, including members of the genera Chlamydomonqs (9), Chlorella (16), Oxytricha (15), Paramecium (2), and Tetrahymena (9). DNA in the germ line micronucleus in Tetrahymena thermophila is unmethylated, while 0.8% of adenines in DNA of the somatic, polyploid macronucleus are modified to N6MeA (3). The function of N6MeA in T.thermophila is not known.During sexual reproduction, the old macronucleus is destroyed and a new macronucleus develops from a mitotic product of the zygotic micronucleus. De novo methylation of DNA in the developing macronucleus is not random but occurs according to a specific pattern. Some GATC sites, for example, are unmethylated while others are methylated in all macronuclear DNA molecules (6). Still others are methylated in a fraction of the molecules that is characteristic of a particular site and that is consistent between cell lines (1, 17; Capowski et al., submitted for publication). Because T. thermophila undergoes phenotypic assortment (for a review, see reference 13), the existence of partially methylated sites implies that there is a methylase in vegetative cells which recognizes unmethylated DNA as a substrate.Methylation of replicating DNA. If de novo methylation contributes to maintenance of the methylation pattern, then both the newly synthesized strand and, to a lesser extent, the parental strand are expected to undergo methylation in replicating DNA. Vegetatively growing cells were cultured in axenic medium containing 1% proteose peptone (4) to a density of 3 x 105 cells per ml. Bromodeoxyuridine (BUdR; 50 ,ug/ml) and [methyl-3H]thymidine (55 to 80 Ci/mmol; concentration, 0.5 to 1.0 mCi/ml) were added to the medium, and the cells were grown for an additional 270 min (1.5 generations). Nuclear DNA was purified as described previously (6). The parental and daughter strands of the newly replicated DNA were separated on alkaline CsCl2-Cs2SO4 gradients (12) with 3.9 g of CsCl2 in each 4.8-ml gradient.After BUdR was incorporated into the DNA and the DNA was labeled with [3H]thymidine, approximately 40% of the DNA, which was assayed by A260 determination, migrated as a broad peak with a heavy density (Fig. la). The majority (92%) of newly replicated DNA, which was labeled with [3H]thymidine, migrated in the gradient as a heavy peak.Thus, replicating DNA was efficiently labeled and there was