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.     

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.     

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.     

De novo cytosine methylation in the differentiating macronucleus of the stichotrichous ciliate Stylonychia lemnae

Dramatic DNA reorganization and elimination processes occur during macronuclear differentiation in ciliates. In this study we analyzed whether cytosine methylation of specific sequences plays a functional role during DNA rearrangement. Three classes of sequences, macronuclear-destined sequences (MDSs, pCE7), members from a large family of transposon-like elements and micronuclear-specific sequences (pLJ01), differing in their structure and future destiny during nuclear differentiation, were studied in the micronucleus, the developing macronucleus and, when present, in the mature macronucleus. While the MDSs become processed to a 1.1 and 1.3 kb gene-sized macronuclear DNA molecule, the family of transposon-like elements represented by MaA81 becomes removed late in the course of polytene chromosome formation. The micronuclear-specific sequence pLJ01 is eliminated together with bulk micronuclear DNA during degradation of polytene chromosomes. No methylated cytosine could be detected in the vegetative macronucleus and no difference in methylation pattern was observed either between micronucleus and developing macronucleus in MDSs or in a micronuclear-specific sequence. However, a significant percentage of the cytosines contained in the transposon-like element becomes methylated de novo in the course of macronuclear differentiation. This is the first demonstration that cytosine methylation in specific sequences occurs during macronuclear differentiation and may provide a first step towards understanding epigenetic factors involved in DNA processing.     

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.     

A mutation in Paramecium tetraurelia reveals functional and structural features of developmentally excised DNA elements.

The excision of internal eliminated sequences (IESs) from the germline micronuclear DNA occurs during the differentiation of a new macronuclear genome in ciliated protozoa. In Paramecium, IESs are generally short (28-882 bp), AT rich DNA elements that show few conserved sequence features with the exception of an inverted-terminal-repeat consensus sequence that has similarity to the ends of mariner/Tcl transposons (KLOBUTCHER and HERRICK 1995). We have isolated and analyzed a mutant cell line that cannot excise a 370-bp IESs (IES2591) from the coding region of the 51A variable surface protein gene. A single micronuclear C to T transition within the consensus sequence prevents excision. The inability to excise IES259 I has revealed a 28-bp IES inside the larger IES, suggesting that reiterative integration of these elements can occur. Together, the consensus sequence mutation and the evidence for reiterative integration support the theory that Paramecium IESs evolved from transposable elements. Unlike a previously studied Paramecium IES, the presence of this IES in the macronucleus does not completely inhibit excision of its Mild-type micronuclear copy through multiple sexual generations.     

Genomic Organization and Developmental Fate of Adjacent Repeated Sequences in a Foldback DNA Clone of Tetrahymena thermophila

DNA sequence elimination and rearrangement occurs during the development of somatic cell lineages of eukaryotes and was first discovered over a century ago. However, the significance and mechanism of chromatin elimination are not understood. DNA elimination also occurs during the development of the somatic macronucleus from the germinal micronucleus in unicellular ciliated protozoa such as Tetrahymena thermophila. In this study foldback DNA from the micronucleus was used as a probe to isolate ten clones. All of those tested (4/4) contained sequences that were repetitive in the micronucleus and rearranged in the macronucleus. The presence of inverted repeated sequences was clearly demonstrated in one of them by electron microscopy. DNA sequence analysis showed that the left portion of this clone contains three tandem, directly repeated copies of a 340-bp sequence, a 120-bp portion of which appears in inverted orientation at a 1.6-kb distance. This clone, pTtFB1, was subjected to a detailed analysis of its developmental fate. Subregions were subcloned and used as probes against Southern blots of micronuclear and macronuclear DNA. We found that all subregions defined repeated sequence families in the micronuclear genome. A minimum of four different families was defined, two of which are retained in the macronucleus and two of which are completely eliminated. The inverted repeat family is retained with little rearrangement. Two of the families, defined by subregions that do not contain parts of the inverted repeat, one in the "loop" and one in the "right flanking region," are totally eliminated during macronuclear development--and contain open reading frames. A fourth family occurs in the "loop" region and is rearranged extensively during development. The two gene families that are eliminated are stable in the micronuclear genome but are not clustered together as evidenced by experiments in which DNAs from nullisomic strains are used to map family members to specific micronuclear chromosomes. The inverted repeat family is also stable in the micronuclear genome and is dispersed among several chromosomes. The significance of retained inverted repeats to the process of elimination is discussed.     

Micronuclear DNA of Oxytricha nova contains sequences with autonomously replicating activity in Saccharomyces cerevisiae.

Oxytricha nova is a hypotrichous ciliate with micronuclei and macronuclei. Micronuclei, which contain large, chromosomal-sized DNA, are genetically inert but undergo meiosis and exchange during cell mating. Macronuclei, which contain only small, gene-sized DNA molecules, provide all of the nuclear RNA needed to run the cell. After cell mating the macronucleus is derived from a micronucleus, a derivation that includes excision of the genes from chromosomes and elimination of the remaining DNA. The eliminated DNA includes all of the repetitious sequences and approximately 95% of the unique sequences. We cloned large restriction fragments from the micronucleus that confer replication ability on a replication-deficient plasmid in Saccharomyces cerevisiae. Sequences that confer replication ability are called autonomously replicating sequences. The frequency and effectiveness of autonomously replicating sequences in micronuclear DNA are similar to those reported for DNAs of other organisms introduced into yeast cells. Of the 12 micronuclear fragments with autonomously replicating sequence activity, 9 also showed homology to macronuclear DNA, indicating that they contain a macronuclear gene sequence. We conclude from this that autonomously replicating sequence activity is nonrandomly distributed throughout micronuclear DNA and is preferentially associated with those regions of micronuclear DNA that contain genes.     

Internal micronuclear DNA regions which include sequences homologous to macronuclear telomeres are deleted during development in Tetrahymena

C4A2 repeats are present in multiple clusters in both the macronucleus and micronucleus of Tetrahymena. Although the macronucleus is generated from the micronucleus after sexual conjugation, the repeats are telomeric sequences in the macronucleus but are internally located in the micronucleus (1). This study investigates the fate of the sequences adjacent to the micronuclear C4A2 repeats. Southern blot analyses of 21 C4A2-containing micronuclear clones show that extensive elimination of the adjacent sequences occurs during the formation of the macronucleus. Comparison of one C4A2-containing micronuclear clone with its derived macronuclear segment indicates that approximately 4.5 kb of DNA, which includes the C4A2 repeats and adjacent sequences on both sides is deleted from the macronucleus. The two regions adjoining the deletion are joined together to form a contiguous segment in the macronucleus. This excision of C4A2 repeats and surrounding sequences and the rejoining of the retained segments is probably the mechanism by which all or most of the other C4A2 adjacent sequences are eliminated.     

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.     

The processing of macronuclear-destined DNA sequences microinjected into the macronuclear anlagen of the hypotrichous ciliate Stylonchia lemnae.

We describe the construction of a vector carrying the micronuclear versions of two macronuclear DNA molecules, one of which was modified by the insertion of a polylinker sequence. This vector was injected into the polytene chromosomes of the developing macronucleus of Stylonychia and its processing during further macronuclear development and its fate in the mature macronucleus were analyzed. In up to 30% of injected cells the modified macronuclear DNA sequence could be detected. While the internal eliminated sequences (IES) present in the macronuclear precursor DNA sequence are still retained in the mature macronucleus, the modified macronuclear DNA sequence is correctly cut out from the vector, telomeres are added de novo and it is stably retained in the macronucleus during vegetative growth of the cells. This vector system represents an experimental system that allows the identification of DNA sequences involved in the processing of macronuclear DNA sequences during macronuclear development.     

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 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.     

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.     

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.     

Analysis of micronuclear, macronuclear and cDNA sequences encoding the regulatory subunit of cAMP-dependent protein kinase of Euplotes octocarinatus: evidence for a ribosomal frameshift

We have isolated and characterized the micronuclear gene encoding the regulatory subunit of cAMP-dependent protein kinase of the ciliated protozoan Euplotes octocarinatus, as well as its macronuclear version and the corresponding cDNA. Analyses of the sequences revealed that the micronuclear gene contains one small 69-bp internal eliminated sequence (IES) that is removed during macronuclear development. The IES is located in the 5'-noncoding region of the micronuclear gene and is flanked by a pair of tetranucleotide 5'-TACA-3' direct repeats. The macronuclear DNA molecule carrying this gene is approximately 1400 bp long and is amplified to about 2000 copies per macronucleus. Sequence analysis suggests that the expression of this gene requires a +1 ribosomal frameshift. The deduced protein shares 31% identity with the cAMP-dependent protein kinase type I regulatory subunit of Homo sapiens, and 53% identity with the regulatory subunit R44 of one of the two cAMP-dependent protein kinases of Paramecium. In addition, it contains two highly conserved cAMP binding sites in the C-terminal domain. The putative autophosphorylation site ARTSV of the regulatory subunit of E. octocarinatus is similar to that of the regulatory subunit R44 of Paramecium but distinct from the consensus motif RRXSZ of other eukaryotic regulatory subunits of cAMP-dependent protein kinases.     

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.