Differential DNA Amplification and Copy Number Control in the Hypotrichous Ciliate Euplotes crassus

ABSTRACT. During macronuclear development in hypotrichous ciliated protozoans, several thousand macronuclear DNA molecules are amplified several-hundred fold. We investigated the regulation of this amplification by determining the copy numbers of three different macronuclear DNA molecules in the hypotrichous ciliate Euplotes crassus. Two of the macronuclear DNA molecules were present in approximately 1,000 copies per cell, while the third was present in approximately 6,500 copies per cell. These reiteration levels were achieved either during macronuclear development, or shortly thereafter, and were maintained during vegetative growth. The most abundant macronuclear DNA molecule is present as a single-copy sequence in the micronuclear genome. Thus, its high copy number results from differential amplification. These results indicate that DNA amplification during macronuclear development is regulated individually for each macronuclear DNA molecule.     

Large scale synchronous mating and the study of macronuclear development in Euplotes crassus

After conjugation in hypotrichous ciliates, a new macronucleus is produced from a copy of the micronucleus. This transformation involves large-scale reorganization of DNA, with conversion of the chromosomal micronuclear genome into short, gene-sized DNA molecules in the macronucleus. To study directly the changes that occur during this process, we have developed techniques for synchronous mating of large populations of the hypotrichous ciliate Euplotes crassus. Electron microscope studies show that the micronuclear chromosomes are polytenized during the first 20 h of macronuclear development. The polytene chromosomes lack the band-interband organization observed in other hypotrichs and in the Diptera. Polytenization is followed by transectioning of the chromosomes. We isolated DNA at various times of macronuclear development and found that the average molecular weight of the DNA decreases at the time of chromosome transectioning. In addition, we have shown that a small size group of macronuclear DNA molecules (450-550 base pairs) is excised from the chromosomal DNA approximately 10 h later in macronuclear development.     

The DNA of ciliated protozoa.

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons.     

The DNA of ciliated protozoa.

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons.     

Internal sequences are eliminated from genes during macronuclear development in the ciliated protozoan Oxytricha nova

During the life cycle of the hypotrichous ciliate Oxytricha nova, a macronucleus containing short, gene-sized DNA molecules is produced from a copy of the chromosomal micronuclear genome. In order to characterize the process of macronuclear development, we have isolated and determined the DNA sequence of a particular macronuclear gene and its micronuclear precursor. The results of this analysis indicate that macronuclear telomeric sequences (5'C4A4(3') repeats) are not present at the ends of the gene in its micronuclear chromosomal location and must be added during development. In addition, the micronuclear copy of the gene contains three short blocks of sequence that must be removed during development, implying the involvement of a nucleic acid-splicing process in generating mature macronuclear genes.     

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.     

Alternative use of chromosome fragmentation sites in the ciliated protozoan Oxytricha nova.

During its life cycle, the hypotrichous ciliated protozoan Oxytricha nova transforms a copy of its micronucleus, which contains chromosome-sized DNA, into a macronucleus containing linear, gene-sized DNA molecules. A region of the micronuclear genome has been defined that gives rise to two distinct macronuclear DNA molecules during development. Through analysis of recombinant macronuclear and micronuclear clones, the generation of the two macronuclear DNA molecules was shown to be the result of alternative use of chromosome fragmentation sites. In addition, evidence was obtained that adjacent micronuclear precursors of macronuclear DNA molecules can overlap by a few base pairs. The significance of these findings in relation to developmental chromosome fragmentation is discussed.     

Variable copy number of macronuclear DNA molecules in Tetrahymena.

In Tetrahymena, the DNA of the macronucleus exists as very large (100 to 4,000-kb) linear molecules that are randomly partitioned to the daughter cells during cell division. This genetic system leads directly to an assortment of alleles such that all loci become homozygous during vegetative growth. Apparently, there is a copy number control mechanism operative that adjusts the number of each macronuclear DNA molecule so that macronuclear DNA molecules (with their loci) are not lost and aneuploid death is a rare event. In comparing Southern analyses of the DNA from various species of Tetrahymena using histone H4 genes as a probe, we find different band intensities in many species. These differences in band intensities primarily reflect differences in the copy number of macronuclear DNA molecules. The variation in copy number of macronuclear DNA molecules in some species is greater than an order of magnitude. These observations are consistent with a developmental control mechanism that operates by increasing the macronuclear copy number of specific DNA molecules (and the genes located on these molecules) to provide the relatively high gene copy number required for highly expressed proteins.     

Macronuclear gene-sized molecules of hypotrichs.

The macronuclear genome of hypotrichous ciliates consists of DNA molecules of gene-sized length. A macronuclear DNA molecule contains a single coding region. We have analyzed the many hypotrich macronuclear DNA sequences sequenced by us and others. No highly conserved promoter sequences nor replication initiation sequences have been identified in the 5' nor in the 3' non-translated regions, suggesting that promoter function in hypotrichs may differ from other eukaryotes. The macronuclear genes are intron-poor; approximately 19% of the genes sequenced to date have one to three introns. Not all macronuclear DNA molecules may be transcribed; some macronuclear molecules may not have any coding function. Codon bias in hypotrichs is different in many respects from other ciliates and from other eukaryotes.     

Variable copy number of macronuclear DNA molecules in Tetrahymena

In Tetrahymena, the DNA of the macronucleus exists as very large (100 to 4,000-kb) linear molecules that are randomly partitioned to the daughter cells during cell division. This genetic system leads directly to an assortment of alleles such that all loci become homozygous during vegetative growth. Apparently, there is a copy number control mechanism operative that adjusts the number of each macronuclear DNA molecule so that macronuclear DNA molecules (with their loci) are not lost and aneuploid death is a rare event. In comparing Southern analyses of the DNA from various species of Tetrahymena using histone H4 genes as a probe, we find different band intensities in many species. These differences in band intensities primarily reflect differences in the copy number of macronuclear DNA molecules. The variation in copy number of macronuclear DNA molecules in some species is greater than an order of magnitude. These observations are consistent with a developmental control mechanism that operates by increasing the macronuclear copy number of specific DNA molecules (and the genes located on these molecules) to provide the relatively high gene copy number required for highly expressed proteins. © 1992 Wiley-Liss, Inc.     

Gene-sized macronuclear DNA molecules are clustered in micronuclear chromosomes of the ciliate Oxytricha nova.

Following the sexual phase of its life cycle, the hypotrichous ciliate Oxytricha nova transforms a copy of its chromosomal micronucleus into a macronucleus containing short, linear DNA molecules with an average size of 2.2 kilobase pairs. In addition, more than 90% of the DNA sequences in the micronuclear genome are eliminated during this process. We have examined the organization of macronuclear DNA molecules in the micronuclear chromosomes. Macronuclear DNA molecules were found to be clustered and separated by less than 550 base pairs in two cloned segments of micronuclear DNA. Recombinant clones of two macronuclear DNA molecules that are adjacent in the micronucleus were also isolated and examined by DNA sequencing. The two macronuclear DNA molecules were found to be separated by only 90 base pairs in the micronuclear genome.     

Genetic characterization and use of a restriction fragment length variant in the hypotrichous ciliate Euplotes crassus

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.     

DNA intermediates and telomere addition during genome reorganization in Euplotes crassus

Three gene-sized molecules cloned intact from macronuclear DNA served as hybridization probes to study excision of these molecules from chromosomes and their processing during macronuclear development in the hypotrich Euplotes crassus. These molecules occur in integrated forms within polytene chromosomal DNA during macronuclear developmental. After transection of the polytene chromosomes, the three molecules occur in intermediate forms. One of the three molecules first appeared in a large intermediate that was subsequently replaced by a second intermediate, approximately 140 bp larger than the final molecule. The other two macronuclear molecules were detected only in intermediates approximately 140 bp larger than the mature form. These penultimate intermediates are larger by virtue of oversized telomeres, which are pared to yield the mature gene-sized molecules.     

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

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.     

Characterization of a family of repetitive sequences that is eliminated late during macronuclear development of the hypotrichous ciliate Stylonychia lemnae

A repetitive element from the hypotrichous ciliate Stylonychia lemnae was characterized by restriction and hybridization analysis. This repetitive element is present in about 5,000–7,000 copies per haploid genome in the micronucleus and the macronuclear anlagen. Its DNA sequence is very conserved, but the length of the repetitive sequence blocs is variable. In some cases, it is associated with telomeric sequences and macronucleus–homologous sequences. Restriction analysis of genomic micronuclear and macronuclear anlagen DNA and in situ hybridization showed that the repetitive sequences are amplified during the formation of polytene chromosomes. They are localized in many bands of the polytene chromosomes and are eliminated during the degradation of the polytene chromosomes. Possible functions of the repetitive sequences during macronuclear differentiation are discussed. Dev. Genet. 21:201–211, 1997.© 1997 Wiley-Liss, Inc.     

Alteration of the Tetrahymena Genome During Nuclear Differentiation*†

Synopsis.
The DNA of the macro- and the micronucleus of Tetrahymena thermophila has been compared by various biochemical methods. It became evident from their thermal denaturation temperatures and buoyant densities that the 2 DNAs were very similar in overall composition. Small differences were detected when the sequence complexities of these DNAs were compared by DNA renaturation studies. The studies suggested that ˜ 10% of the micronuclear genome was lost or underrepresented in the macronucleus. Comparison of individual gene levels revealed further differences. By using the technic of gene cloning a micronuclear sequence was isolated which hybridized only with micronuclear, but not with macronuclear DNA. These results indicated the occurrence of elimination or underreplication of this sequence in the macronucleus. Gene amplification was also shown to occur. In the micronucleus only a single copy of rDNA was found integrated into the chromosome. During macro-nuclear development, amplification was observed to occur, and the amount of rDNA to increase, until there were ˜ 200 copies per haploid genome in the mature macronucleus. all of them extrachromosomal and palindromic. The 3rd case of alteration involved a simple repeated sequence, (CCCCAA)n, present in the termini of rDNA and also in many other locations of the genome. Restriction endonuclease digestion studies revealed drastic differences in the organization of the repeats between macro-and micronucleus. These differences may be interpreted as the results of chromosome fragmentation which occurs at every cluster of the repeats during macronuclear development. The relationship between this event and gene amplification and elimination is discussed.