Genetic Characterization and Use of a Restriction Fragment Length Variant in the Hypotrichous Ciliate Euplotes crassus¹

Abstract

Two forms of a macronuclear DNA molecule differing in the presence or absence of a restriction endonuclease recognition site have been detected in the hypotrichous ciliate Euplotes crassus. Through a series of genetic crosses the two forms were shown to be allelic, being derived from a single micronuclear genetic locus. This restriction fragment length variant (RFLV) was used as a genetic marker to determine that the migratory and stationary pronuclei generated during mating can be genetically non-identical. In addition, the RFLV was used to investigate the efficiency of processing of the alternate alleles during macronuclear development and their subsequent transmission during vegetative growth. Little or no bias in the processing and/or amplification of the two alleles was observed during macronuclear development. During vegetative growth, however, changes in the relative amounts of the two alleles were observed.

RNA-mediated epigenetic regulation of DNA copy number

“…The combination of imprecise nuclear division during vegetative growth and high ploidy allows individual chromosome copy number to vary widely (26,27). In addition, most reports of DNA copy number in ciliates reflect population averages that mask individual variation, if copy number is not measured in individual cells.…”

“…Although longer, germline chromosomes participate in conventional mitosis during vegetative growth, the tiny somatic macronuclear chromosomes that differentiate from the germline chromosomes undergo amitosis, permitting both drift and regulation of copy number (26,27). Observed levels vary over four orders of magnitude (23,25,(27)(28)(29)(30)(31). With such high copy numbers, a typical macronucleus may contain tens of millions of nanochromosomes, averaging 2.2 kb bound by short telomeres (23).…”

Precise excision of telomere-bearing transposons during Oxytricha fallax macronuclear development.

“…If imprecise excisions occurred, many would have left the gene inoperative, and selection may have weeded out cells with a significant fraction of their chromosomes imprecisely constructed at that site. Because macronuclear genome composition is uniquely flexible, as a result of the phenomenon of macronuclear assortment, evolution of pure macronuclear genotypes from initially heterogeneous ones is to be expected, especially if a selection exists (Baird and Klobutcher 1988;for review, see Brunk 1986;Bruns 1986). Thus, our data strongly demonstrate that precise excision of TBEI-1 and TBE1-2 does occur in macronuclear development, but they allow the possibility that excision is variable, sometimes being imprecise, but at least sometimes precise.…”

Developmentally excised DNA sequences in Euplotes crassus capable of forming G quartets.

“…E. crassus culture and matings were as described (24), with minor modifications (4,11). Strains used for matings were either ST9 and ST11 or CC100 and 14 (9,11,25). Developing macronuclei were purified and DNA was isolated as described (5,24).…”

“…Both are bounded by a direct repeat of the dinucleotide 5'-TA-3', and excision has been shown to be precise such that one copy of the 5'-TA-3' repeat is retained in the resulting macronuclear DNA molecule (11). The excision process occurs during the polytene chromosome stage of development, with the Tec elements being excised early in the polytenization process (25)(26)(27)(28)(29)(30) The current study presents evidence for a class of IESs in E. crassus termed telomeric-repeat-like IESs* (TelIESs). These IESs differ from the well studied IESs in that they are short (<50 bp), their sequences resemble the telomeric repeats of macronuclear DNA molecules, and they are excised later than the larger IESs.…”