Organization of the Euplotes crassus Micronuclear Genome1

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.     

Macronuclear and micronuclear configurations of a gene encoding the protein synthesis elongation factor EF 1α in Stylonychia lemnae

The micronuclear and macronuclear configurations of a gene encoding the protein synthesis elongation factor EF 1 alpha in the hypotrich ciliate Stylonychia lemnae were compared. The two sequences are generally colinear. The coding sequence of the micronuclear gene is, however, interrupted by a 64 bp insert flanked by a 2 bp direct repeat in a gene region which is moderately conserved among EF 1 alpha genes of different organisms. The insertion site is distinct from known intron positions in eukaryotic EF 1 alpha genes. The insert sequence shows inverted repeats at its ends and thus exhibits typical features of an internal eliminated sequence (IES). Comparison with other such sequences in the related organism Oyxtricha nova shows that the IES falls into a new group of such elements. The macronuclear gene exhibits a strikingly limited codon usage, which cannot be simply explained by the overall base composition of the DNA but probably also relates to the very high copy number of the macronuclear gene and the putative high amount of the gene product.     

Multiple Sequence Versions of the Oxytricha fallax 81-MAC Alternate Processing Family1

The 81-MAC family consists of three sizes of macronuclear chromosomes in Oxytricha fallax. Clones of these and of micronuclcar homologs have been classified according to DNA sequence into three highly homologous (95.9–97.9%), but distinct versions. Version A is represented by a micronuclear clone and by clones of two different-sized macronuclear chromosomes, showing that alternate processing of micronuclear DNA is responsible for the variety of sizes of macronuclcar chromosomes. Three Internal Eliminated Sequences (IES's) are demonstrated in Version A micronuclcar DNA. Two have been sequenced and show short, flanking direct repeats but no inverted terminal repeats. Version C micronuclear DNA has interruptions in the macronuclear homology which correspond closely to the Version A IES's. Whether they are true IES's is unknown because no Version C macronuclear DNA has been demonstrated. Version C micronuclear DNA may be “macronuclear-homologous” but “micronucleus-limited” and not “macronucleusdestined.” Version B is represented by macronuclear DNA clones, but no micronuclear clones. Vegetative micronuclear aneuploidy is suggested. The possible role of micronuclear defects in somatic karyonidai senescence is discussed in light of the precise macronuclear chromosome copy controls demonstrated within the 81-MAC family. These controls apparently operate throughout karyonidai life to mairitain 1) a constant absolute amount of 81-MAC sequences in the macronuclcus and 2) a constant sioichiometry within the family, both according to version and chromosome size.     

Organization of gene and non-gene sequences in micronuclear DNA of Oxytricha nova.

In order to study the derivation of the macronuclear genome from the micronuclear genome in Oxytricha nova micronuclear DNA was partially digested with EcoRI, size fractionated, and then cloned in the lambda phage Charon 8. Clones were selected a) at random b) by hybridization with macronuclear DNA or c) by hybridization with clones of macronuclear DNA. One group of these clones contains only unique sequence DNA, and all of these had sequences that were homologous to macronuclear sequences. The number of macronuclear genes with sequences homologous to these micronuclear clones indicates that macronuclear sequences are clustered in the micronuclear genome. Many micronuclear clones contain repetitive DNA sequences and hybridize to numerous EcoRI fragments of total micronuclear DNA, yielding similar but non-identical patterns. Some micronuclear clones containing these repetitive sequences also contained unique sequence DNA that hybridized to a macronuclear sequence. These clones define a major interspersed repetitive sequence family in the micronuclear genome that is eliminated during formation of the macronuclear genome.     

Conserved DNA sequences adjacent to chromosome fragmentation and telomere addition sites in Euplotes crassus.

During the formation of a new macronucleus in the ciliate Euplotes crassus, micronuclear chromosomes are reproducibly broken at approximately 10 000 sites. This chromosome fragmentation process is tightly coupled with de novo telomere synthesis by the telomerase ribonucleoprotein complex, generating short linear macronuclear DNA molecules. In this study, the sequences of 58 macronuclear DNA termini and eight regions of the micronuclear genome containing chromosome fragmentation/telomere addition sites were determined. Through a statistically based analysis of these data, along with previously published sequences, we have defined a 10 bp conserved sequence element (E-Cbs, 5'-HATTGAAaHH-3', H = A, C or T) near chromosome fragmentation sites. The E-Cbs typically resides within the DNA destined to form a macronuclear DNA molecule, but can also reside within flanking micronuclear DNA that is eliminated during macronuclear development. The location of the E-Cbs in macronuclear-destined versus flanking micronuclear DNA leads us to propose a model of chromosome fragmentation that involves a 6 bp staggered cut in the chromosome. The identification of adjacent macronuclear-destined sequences that overlap by 6 bp provides support for the model. Finally, our data provide evidence that telomerase is able to differentiate between newly generated ends that contain partial telomeric repeats and those that do not in vivo.     

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.     

Invention and Mystery in Hypotrich DNA1

ABSTRACT “The capacity to blunder slightly is the real marvel of DNA. Without this special attribute, we would still be anaerobic bacteria and there would be no music.” Lewis Thomas³ Hypotrichs have evolved extraordinary ways of organizing, manipulating, and replicating the DNA in their micronuclear and macronuclear genomes. Short macronuclear DNA molecules containing single genes are created by excision from chromosomes, accompanied by massive elimination of the germline DNA sequences between genes. Germline genes themselves are interrupted by multiple noncoding segments called internal eliminated segments, or IESs, that divide genes into multiple macronuclear-destined segments, or MDSs. The functional significance of this organization is unknown. Over evolutionary time IESs accumulate mutations rapidly are inserted into or excised from genes, and shift position along DNA molecules. MDSs are ligated to create functional genes when IESs are spliced out of micronuclear DNA during macronuclear development. MDSs in some germline genes are in scrambled disorder and become unscrambled in association with IES elimination. Replication of DNA in the macronucleus is accomplished by organization of replication enzymes and factors into a structure that sweeps through the macronucleus to replicate the many millions of gene-sized DNA molecules. The significance of many of the bizarre DNA phenomena in the evolutionary/functional success of hypotrichs is still unclear.     

The Micronuclear Gene Encoding β-Telomere Binding Protein in Oxytricha nova

ABSTRACT The micronuclear version of the gene encoding β-telomere binding protein (β-TBP) in Oxytricha nova has been sequenced and compared to the macronuclear β-TBP gene, previously described. The micronuclear gene contains three AT-rich internal eliminated sequences (IES) of 37, 40, and 43 bp and four macronuclear destined sequences (MDS). The IES interrupt the gene once near the 5′ end of the coding region and twice in the 3′ trailer downstream from the TGA stop codon. The sequences of the micronuclear and macronuclear genes are colinear. Thus, the micronuclear β-TBP gene is not scrambled, which contrasts with the highly scrambled state among the 14 MDS in the micronuclear α;-TBP gene.     

The micronuclear gene encoding a putative aminoacyl-tRNA synthetase cofactor of the ciliate Euplotes octocarinatus is interrupted by two sequences that are removed during macronuclear development.

The micronuclear gene of the ciliated protozoan Euplotes octocarinatus (Eo) syngen 1 encoding the putative aminoacyl-tRNA synthetase cofactor (ARCE), as well as its macronuclear version and the corresponding cDNA, were amplified and sequenced. Analyses of the sequences revealed that the micronuclear gene contains two sequences (430 and 625bp long) that are missing in the macronuclear version of this gene. These sequences are called 'internal eliminated sequences' (IESs) and appear to occur in all ciliates. The two IESs are located in the coding region of the micronuclear gene. One IES is flanked by a pair of dinucleotide 5'-TA-3' direct repeats and the other one by a pair of hepta-nucleotide 5'-TTACTGA-3' direct repeats. Inside the two IESs, several other sequence repeats were found. The macronuclear DNA molecule carrying this gene is 1517bp long and shows characteristics typical of macronuclear chromosomes of hypotrichous ciliates. Copy number determination revealed that the molecule is amplified to only about 750 copies per macronucleus. The deduced protein is a 441-amino-acid (aa) polypeptide with a molecular mass of 50kDa. It shares a conserved endothelial monocyte-activating polypeptide II (EMAP II)-like carboxyl-terminal domain and a hydrophilic central domain containing a KEKE-motif with a group of proteins associated with aminoacyl-tRNA synthetases and tRNAs.     

Analysis of the micronuclear B type surface protein gene in Paramecium tetraurelia.

The micronuclear DNA of Paramecium contains sequences that are precisely excised during the formation of the macronuclear (somatic) genome. In this paper we show that four eliminated sequences ranging in size from 28 to 416 base pairs, are present in or near the micronuclear copy of the B surface protein gene. Each excised sequence is bounded by the dinucleotide 5'-TdA-3'. Comparison of the micronuclear B gene with the previously determined micronuclear sequence of the A surface protein gene shows that although the positions of at least three of the eliminated sequences are conserved in both genes, the sequences are highly divergent. Transformation of vegetative macronuclei with fragments of the micronuclear B gene results in replication and maintenance of the DNA, but the micronuclear specific sequences are not removed. Previous studies have shown that the correct incorporation of the B gene into the new macronucleus requires copies of the macronuclear B gene in the old macronucleus. Using macronuclear transformation, we show that the micronuclear B gene can substitute for the macronuclear B gene with regard to its role in DNA processing. This suggests that the macronuclear DNA is not acting as a guide for the excision of the micronuclear specific sequences.     

Elimination of germ-line tandemly repeated sequences from the somatic genome of the ciliate Oxytricha fallax

The ciliated protozoa exhibit nuclear dimorphism. The genome of the somatic macronucleus arises from the germ-line genome of the micronucleus following conjugation. We have studied the fates of highly repetitious sequences in this process. Two cloned, tandemly repeated sequences from the micronucleus of Oxytricha fallax were used as probes in hybridizations to micronuclear and macronuclear DNA. The results of these experiments show: (1) the cloned repeats are members of two apparently unrelated repetitious sequence families, which each appear to comprise a few percent of the micronuclear genome, and (2) the amount of either family in the macronuclei from which our DNA was prepared is about 1/15 that found in an equal number of diploid micronuclei. Most, if not all, of the apparent macronuclear copies of these repeats can be accounted for by micronuclear contamination, which strongly suggests that these sequences are eliminated from the macronuclei and have no vegetiative function.     

Characterization of Two Sibling Species of the Genus Stylonychia (Ciliata,Hypotricha): S. mytilus Ehrenberg, 1838 and S. lemnae n. sp. II. Biochemical Characterization1

The two closely related hypotrichous ciliate species, Stylonychia lemnae and S. mytilus, have been compared with respect to their isoenzyme patterns, their macronuclear DNA banding patterns, and micronuclear DNA banding patterns after restriction enzyme digestion. Since macronuclear DNA contains mainly protein-coding sequences and since the micronuclear DNA patterns represent mainly the repetitive fraction of the genome, these results, together with the isoenzyme patterns, reflect differences on three different levels of molecular evolution between morphologically very similar species. Each of the three methods allows an unequivocal identification of each of the two species. Intraspecific variation seems to be greatest among the repetitive sequences of the micronuclear genomes. By using the isoenzyme data and the formula of Nei (19) the genetic distance between the two species is calculated and compared with the results from other protozoa and different Drosophila species. Despite their morphological similarity, the two species show a considerable amount of evolutionary divergence on the three molecular levels which have been investigated.     

Cell cycle mutation in Paramecium tetraurelia discriminates between sexual and vegetative functions

The temperature-sensitive mutation cc1 blocks a number of cell cycle processes in Paramecium including macronuclear DNA synthesis, oral morphogenesis, and the later stages of micronuclear mitosis. Oral morphogenesis and micronuclear mitosis also occur in the sexual pathway. This study shows that cc1 cells can proceed through conjugation or autogamy under restrictive conditions; neither stomatogenesis nor micronuclear mitosis is blocked. Fertilization and macronuclear determination occur normally, but DNA synthesis in macronuclear anlagen is blocked. Therefore, this mutation discriminates between oral replacement during meiosis and vegetative prefission stomatogenesis, and between mitotic spindle elongation during the pregamic and postzygotic divisions and spindle elongation during the vegetative cell cycle. These results point to a fundamental regulatory difference between morphogenesis in the vegetative and sexual pathways. © 1994 Wiley-Liss, Inc.     

Cloning and sequence analysis of the micronuclear and macronuclear gene encoding Rab protein of Euplotes octocarinatus

The DNA in a micronucleus undergoes remarkable rearrangements when it develops into a macronucleus after cell mating in the hypotrichous ciliate. A Rab gene was isolated from the macronuclear plasmid mini-library of Euplotes octocarinatus. A micronuclear version of the Rab gene was amplified by polymerase chain reaction (PCR). The macronuclear DNA molecule carrying the Rab gene is 767 bp long and shows characteristics typical of macronuclear chromosomes of hypotrichous ciliates. Three of the five cysteines are encoded by the opal codon UGA. The deduced protein is a 207-amino acid (aa) with a molecular mass of 23 kDa. The protein shares 36% identity with Rab 1 protein of Plasmodium and yeast. Analysis of the sequences indicated that the micronuclear version of the Rab gene contains two internal eliminated sequences, internal eliminated sequence (IES)1 and IES2. IES1 is flanked by a pair of hepta-nucleotide 5'-AAATTTT-3' direct repeats, and IES2 is flanked by 5'-TA-3' direct repeats.     

Organization of the Euplotes crassus micronuclear genome

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.     

Rare internal C4A4 repeats in the micronuclear genome of Oxytricha fallax.

Approximately 20,000 different short, linear, macronuclear DNA molecules are derived from micronuclear sequences of Oxytricha fallax after conjugation. These macronuclear DNAs are terminated at both ends by 20 base pairs of the sequence 5'-dC4A4-3'. Sequences homologous to this repeat (C4A4+) are also abundant in the micronuclear chromosomes, but most reside at their telomeres. Here we show that nontelomeric C4A4 clusters of 20 base pairs or longer exist in only a few hundred copies per micronuclear genome. This demonstrates that nearly none of the 20,000 sequence blocks of micronuclear DNA destined to be macronuclear DNA molecules can be flanked by full-length (20-base pair) C4A4 clusters, and therefore C4A4 repeats must be added to most, if not all, macronuclear telomeres during macronuclear development. Six internal micronuclear C4A4+ loci were cloned, and their structural relationships with macronuclear and micronuclear sequences were examined. The possible origins and functions of these rare, micronuclear internal C4A4 loci are discussed.     

Micronuclear genome organization in Euplotes crassus: a transposonlike element is removed during macronuclear development.

After mating, hypotrichous ciliated protozoa transform a set of their micronuclear chromosomes into thousands of short, linear DNA molecules that form the macronuclear genome. To examine micronuclear genome organization in the hypotrich Euplotes crassus, we have analyzed two cloned segments of micronuclear DNA as well as the macronuclear DNA molecules that are derived from them. E. crassus was found to display a number of features characteristic of other hypotrich genomes, including (i) clustering and close spacing of the precursors of macronuclear DNA molecules, (ii) the frequent occurrence of internal eliminated sequences within macronuclear precursors, (iii) overlapping macronuclear precursors, (iv) lack of telomeric repeats at the ends of macronuclear precursors, and (v) alternative processing of the micronuclear chromosome to yield multiple macronuclear DNA molecules. In addition, a moderately repetitive, transposonlike element that interrupts the precursors of two macronuclear DNA molecules has been identified and characterized. This transposonlike element, designated Tec1, is shown to be reproducibly removed from one of the macronuclear precursors during independent episodes of macronuclear development.     

Common terminal repeats of the macronuclear DNA are absent from the micronuclear DNA in hypotrichous ciliate, Stylonychia pustulata.

Comparison of nucleotide sequences of a macronuclear DNA and its micronuclear counterpart of a hypotrichous ciliate, Stylonychia pustulata, demonstrates that common terminal repeats (C4A4) of the macronuclear DNA are not present at the corresponding region in the micronuclear genome. The results indicate that the common terminal C4A4 repeat is added or translocated during or after the rearrangement of the micronuclear DNA to the macronuclear DNA.     

Multiple sequence versions of the Oxytricha fallax 81-MAC alternate processing family

The 81-MAC family consists of three sizes of macronuclear chromosomes in Oxytricha fallax. Clones of these and of micronuclear homologs have been classified according to DNA sequence into three highly homologous (95.9-97.9%), but distinct versions. Version A is represented by a micronuclear clone and by clones of two different-sized macronuclear chromosomes, showing that alternate processing of micronuclear DNA is responsible for the variety of sizes of macronuclear chromosomes. Three Internal Eliminated Sequences (IES's) are demonstrated in Version A micronuclear DNA. Two have been sequenced and show short, flanking direct repeats but no inverted terminal repeats. Version C micronuclear DNA has interruptions in the macronuclear homology which correspond closely to the Version A IES's. Whether they are true IES's is unknown because no Version C macronuclear DNA has been demonstrated. Version C micronuclear DNA may be "macronuclear-homologous" but "micronucleus-limited" and not "macronucleus-destined." Version B is represented by macronuclear DNA clones, but no micronuclear clones. Vegetative micronuclear aneuploidy is suggested. The possible role of micronuclear defects in somatic karyonidal senescence is discussed in light of the precise macronuclear chromosome copy controls demonstrated within the 81-MAC family. These controls apparently operate throughout karyonidal life to maintain 1) a constant absolute amount of 81-MAC sequences in the macronucleus and 2) a constant stoichiometry within the family, both according to version and chromosome size.