Macronuclear DNA of the hypotrichous ciliate Oxytricha fallax

Abstract

DNA in the macronuclei of Oxytricha fallax, as in other hypotrichous ciliate protozoa, exists as small, achromosomal molecules rather than in chromosomes. We report studies on 0. fallax DNA using physicochemical procedures and nucleic acid hybridization. Macronuclear DNA molecules range in size from 22 kilobase pairs (kb) to about 0.5 kb. The DNA has a buoyant density in CsCl of 1.694 gcm-3 and a melting temperature in 15 mM NaCl/1.5 mM sodium citrate, pH 7, at 65.40. These values correspond to 34.7% Gua + Cyt and 28.1% Gua + Cyt, respectively, and base composition determined by thin-layer chromatography of nucleotides is 32.4% Gua + Cyt. The only modified nucleotide that is detectable is N6-methyldeoxyadenylate (0.2%), and the amount and kind of modification cannot account for the discrepancies in nucleotide composition determination by the three methods. The (rDNA) in the oocytes of amphibia (1, 2) and some insects (3, 4) and in the ciliate protozoa Tetrahymena (5, 6) and Paramecium (7). rDNA is also extrachromosomal in Physarum (8) and Dictyostelium (9). In Tetrahymena macronuclei (10) and in Physarum, rDNA is in the form of palindromic dimers. In Paramecium and other organisms, amplified genes for rRNA are tandemly repeated several to many times in extrachromosomal DNA molecules.Hypotrichous ciliate protozoa such as Oxytricha have a macronuclear genome comprised entirely of "extrachromosomal" or "achromosomal" DNA (11). These DNA pieces are produced by fragmentation of polytene chromosomes in postconjugative cells (12,13), and the process of macronuclear development from micronuclei results in considerable reduction in DNA sequence complexity (14). As was shown by Lawn and coworkers (15), macronuclear DNA is heterodisperse, but a particular DNA sequence in the macronucleus is found in a single size class of DNA molecules or in a small variety of discrete sizes.In this report, characteristics of the achromosomal macronuclear DNA of Oxytricha fallax are presented. 0. fallax is related to the unidentified species studied in Prescott's laboratory (15), but it is distinguished from the latter by its smaller size, by a different DNA buoyant density in CsCl, and by having two micronuclei per cell instead of four. Nevertheless, the processes of conjugation, macronuclear development, and vegetative reproduction in 0. fallax follow the general pattern described for other hypotrichs. Our reported studies on 0. fallax have concentrated on the genes for 25S and 19S rRNAs and for 5S rRNA. These genes have been localized to relatively small, nonpalindromic DNA, little larger than necessary to accomodate single coding sequences.MATERIALS AND METHODS Culture and In Vivo Labeling of 0. fallax. Cultures of 0. fallax were obtained from the Indiana University collection. Cells were grown in 0.1% Cerophyl medium with Chlamydonas reinhardtii as a food source. Medium was supplemented either with heat-killed Escherichia coli or with killed Klebsiella aerogenes in a vitamin/stigmasterol mixture (16). Cultures were harves...

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

“…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.…”

N6-methyladenine: the other methylated base of DNA

“…A critical point for the detection of m6A in mammalian DNA is the sensitivity of the methodologies used. In this regard, it is noteworthy that current data available, which do not detect m6A in mammalian DNA, have been obtained with techniques Enterobacteriales E.coli 1.4-2.0 (2) Mycobacteria smegmatis 3.0 (90) Mycobacteria tuberculosis 0.45-0.5 (90) Mycoplasma arginini 2.0 (91) Mycoplasma capricolum 0.2 (91) Mycoplasma hyorhinis 2.0 (91) Mycoplasma orale 0.9 (91) Acholeplasma laidlawii 0.5 (91) Eucarya Oxytricha fallax 0.6-0.7 (43) Paramecia aurelia 2.5 (14) Chlamydomonas reinhardi 0.50 (41) Tetrahymena pyriformis 0.65-0.80 (41,48) Tetrahymena thermophila 0.80 (44) Viridiplantae, Wheat 0.09-0.10 (mt) (61) 0.46-0.55 (n) (61) Arthropoda, Aedes albopictus 0.1 (63) mt: mitochondrial, n: nuclear. %m6A were calculated on the basis of total Adenine residues.…”

High-Resolution Mapping of N6-Methyladenosine in Transcriptome and Genome Using a Photo-Crosslinking-Assisted Strategy

“…The deletion of 6mA methyltransferase in pathogenic Escherichia coli results in global transcription changes, indicating the likelihood of 6mA not only serving as a host genetic marker but also possessing regulatory functions (Fang et al, 2012). Besides bacteria, certain unicellular eukaryotes also have relatively abundant 6mA in their genomes, for instance, the protozoan Tetrahymena (Harrison, Findly, & Karrer, 1986;Hattman, Kenny, Berger, & Pratt, 1978), Oxytricha fallax (Rae & Spear, 1978), Paramecium aurelia (Cummings, Tait, & Goddard, 1974), and green algae Chlamydomonas reinhardtii (Hattman et al, 1978). However, the lack of methylation-sensitive restriction endonucleases suggests that 6mA possesses specific regulatory functions in these unicellular eukaryotes (Ehrlich & Zhang, 1990).…”