The Euplotes crassus conjugation-specific conN1 gene encodes a transcription elongation factor TFIIS-like protein.

The Euplotes crassus macronuclear DNA molecule containing the conjugation-specific conN1 gene has been sequenced, along with a cDNA clone. The results indicate that the conN1 gene encodes a protein similar to the transcription elongation factor TFIIS proteins identified in other eukaryotes.

conZA8 encodes an abundant protein targeted to the developing macronucleus in Euplotes crassus.

During macronuclear development in the ciliate Euplotes crassus, micronuclear-derived chromosomes undergo a series of rearrangements that include polytenization, DNA splicing, chromosome fragmentation, and telomere addition and processing. Although cis-acting signals that may function in the regulation of these events have been characterized, the proteins that mediate these events have not yet been identified. To identify development-specific factors that may be involved in DNA rearrangement, we previously isolated clones of a number of genes that are expressed only during early macronuclear development. Here, we report the genomic and cDNA sequences of one of these genes, conZA8. The analysis indicates that the conZA8 gene encodes a novel, 468-amino acid, proline-rich protein. Antibodies were raised against both a recombinant form of the conZA8 protein and an internal peptide. Immunoblotting and immunofluorescence analyses indicated that the conZA8 protein is highly abundant, expressed only during the polytene chromosome stage of macronuclear development, and localized to the developing macronucleus. Possible functions of the conZA8 protein are discussed.

A development-specific histone H3 localizes to the developing macronucleus of Euplotes.

During the process of macronuclear development, the ciliate Euplotes crassus undergoes extensive programmed DNA rearrangement. Previous studies have identified a gene, H3(P), that is expressed only during sexual reproduction and is predicted to encode a variant histone H3 protein. In the current study, an antiserum to the H3(P) protein has been generated. The antiserum has been used to demonstrate that H3(P) is maximally expressed during the polytene chromosome stage of macronuclear development. Moreover, H3(P) is localized to the developing macronucleus, but not other nuclei present within the cell. Additional studies indicate that at least one additional variant histone is also present within the developing macronucleus. The results indicate that there are significant changes in nucleosome composition within the developing macronucleus, and provide additional support for the notion that changes in chromatin structure play a role in the DNA rearrangement processes of macronuclear development. genesis 26:179-188, 2000.

Characterization of in vivo developmental chromosome fragmentation intermediates in E. crassus.

Ligation-mediated PCR was used to characterize intermediates in the fragmentation/de novo telomere addition process that occurs during sexual reproduction in the ciliate E. crassus. Fragmentation generates ends with 6-base, 3' overhangs that have 5'-phosphate and 3'-hydroxyl groups. These intermediates are detected only during the period of chromosome fragmentation. Fragmentation always occurs at a precise distance from a conserved sequence, the E-Cbs, indicating that it is a key cis-acting element in the process. The results also serve to identify the natural substrate for de novo telomere addition and indicate that telomerase recognizes, and compensates for, partial telomeric repeats at the ends of fragmentation intermediates. Similarities of the Euplotes fragmentation/telomere addition process to the movement of some non-long terminal repeat retrotransposons are discussed.

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.

An unusual histone H3 specific for early macronuclear development in Euplotes crassus.

Characterization of the histone H3 genes of the ciliated protozoan Euplotes crassus indicates that one gene functions only during the sexual phase of the life cycle. Maximum expression of this gene, as judged by transcript accumulation, correlates with DNA replications leading to polytenization of the micronuclear chromosomes before massive DNA elimination, which produces a transcriptionally active macronucleus. Transcripts of the other gene accumulate primarily during vegetative growth and in the sexual phase of the life cycle during replication phases not related to polytenization. Although both histone H3 genes encode proteins that are fairly divergent in sequence at the amino terminus, the meiotic/polytene-specific histone H3 contains two insertions in the amino terminus that increase the size of the protein by 15 amino acids. Analysis of micrococcal nuclease digests of chromatin using hybridization probes specific for micronuclear vs. macronuclear sequences indicates that a change in nucleosomal spacing correlates with the maximal expression of the meiotic/polytene-specific histone H3 gene. Thus, we surmise that this unusual histone H3 may play a key role in targeting DNA sequences for either transcriptional activation and retention in the macronucleus or heterochromatization and elimination.

Conjugation-specific genes in the ciliate Euplotes crassus: gene expression from the old macronucleus.

Following mating or conjugation, the hypotrichous ciliate Euplotes crassus undergoes a massive genome reorganization process. While the nature of the rearrangement events has been well studied, little is known concerning proteins that carry out such processes. As a means of identifying such proteins, differential screening of a developmental cDNA library, as well as construction of a cDNA subtraction library, was used to isolate genes expressed only during sexual reproduction. Five different conjugation-specific genes have been identified that are maximally expressed early in conjugation, during the period of micronuclear meiosis, which is just prior to macronuclear development and the DNA rearrangement process. All five genes are retained in the mature macronucleus. Micronuclear, macronuclear, and cDNA clones of one gene (conZA7) have been sequenced, and the results indicate that the gene encodes a putative DNA binding protein. In addition, the presence of an internal eliminated sequence in the micronuclear copy of the conZA7 gene indicates that this conjugation-specific gene is transcribed from the old macronucleus.

Micronuclear and macronuclear sequences of a Euplotes crassus gene encoding a putative nuclear protein kinase.

The sequences of a 1.8-kbp macronuclear DNA molecule (V3), and the majority of its micronuclear counterpart, are reported. The macronuclear V3 DNA molecule contains an open reading frame that is interrupted by a single intron, while the micronuclear copy is interrupted by four internal eliminated sequences, one of which is located within the intron. The predicted protein product of the macronuclear V3 gene is a 471-amino acid polypeptide that is very similar to a group of protein-serine/threonine kinases from both plant and animal species, some of whose members appear to be involved in cell cycle or growth control.

Genetic code deviations in the ciliates: evidence for multiple and independent events.

In several species of ciliates, the universal stop codons UAA and UAG are translated into glutamine, while in the euplotids, the glutamine codon usage is normal, but UGA appears to be translated as cysteine. Because the emerging position of this monophyletic group in the eukaryotic lineage is relatively late, this deviant genetic code represents a derived state of the universal code. The question is therefore raised as to how these changes arose within the evolutionary pathways of the phylum. Here, we have investigated the presence of stop codons in alpha tubulin and/or phosphoglycerate kinase gene coding sequences from diverse species of ciliates scattered over the phylogenetic tree constructed from 28S rRNA sequences. In our data set, when deviations occur they correspond to in frame UAA and UAG coding for glutamine. By combining these new data with those previously reported, we show that (i) utilization of UAA and UAG codons occurs to different extents between, but also within, the different classes of ciliates and (ii) the resulting phylogenetic pattern of deviations from the universal code cannot be accounted for by a scenario involving a single transition to the unusual code. Thus, contrary to expectations, deviations from the universal genetic code have arisen independently several times within the phylum.

Consensus inverted terminal repeat sequence of Paramecium IESs: resemblance to termini of Tc1-related and Euplotes Tec transposons.

During the formation of a transcriptionally active macronucleus, ciliated protozoa excise large numbers of interstitial segments of DNA (internal eliminated sequences; IESs) from their chromosomes. In this study we analyze the published sequences of 20 IESs that interrupt surface protein genes of Paramecium and identify a consensus inverted terminal repeat. This sequence is similar to the ends of the Tc1-related transposons found in nematodes and other metazoans, as well as to both the ends of the Tec transposons and at least some of the IESs in the distantly related ciliate Euplotes crassus. The results of these analyses bolster previous proposals that IESs were created by transposition.

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

Tens of thousands of DNA segments are eliminated by DNA breakage and rejoining events during the formation of a new macronucleus in the hypotrichous ciliated protozoan Euplotes crassus. This study presents evidence for a class of eliminated sequences referred to as telomeric-repeat-like internal eliminated sequences (TelIESs). TelIESs are shorter (< 50 bp) than most previously characterized IESs and their DNA sequences resemble the telomeric repeat sequences of the organism. The TelIESs are excised during the developmental period of chromosome fragmentation/telomere addition, which is later than previously characterized IESs. Additional studies demonstrate that oligonucleotides representing the TelIESs are, like telomeric repeats, capable of forming G-quartet structures in vitro.

Characterization of transcription initiation, translation initiation, and poly(A) addition sites in the gene-sized macronuclear DNA molecules of Euplotes.

The DNA in the transcriptionally active macronucleus of the hypotrichous ciliate Euplotes crassus exists as short, linear molecules with each molecule encoding a single genetic function. Previous work has indicated that coding regions occupy the majority of macronuclear DNA molecules. In the present study we have defined the transcription initiation sites and poly(A) addition sites for a number of different macronuclear genes in Euplotes crassus. Our results indicate that mature transcripts represent all but approximately 100-200 bases of the non-telomeric sequences in macronuclear DNA molecules. We have also examined the sequences in the vicinity of transcription start sites, poly(A) addition sites, and translation initiation sites for Euplotes species genes in an attempt to define the cis-acting elements that control these processes. Our results indicate that some of the common sequence elements known to control these processes in higher eukaryotes are likely not utilized by Euplotes genes. The data do indicate the presence of other conserved sequences both preceding and at the site of poly(A) addition, as well as at the site of translation initiation. These conserved sequences may serve an analogous role in these organisms. Finally, we have found that most macronuclear DNA molecules have transcription initiation sites within 30 bp of the telomere, suggesting that the telomere may play a role in promoting transcription.

Circular forms of developmentally excised DNA in Euplotes crassus have a heteroduplex junction.

Extensive DNA elimination via a DNA breakage and rejoining process occurs during macronuclear development in the hypotrich ciliate Euplotes crassus. The excision process involves the removal of short, unique segments of DNA (internal eliminated sequences; IESs) and at least two highly repetitive families of transposon-like elements (Tec elements). Previous studies have demonstrated that circular forms of both IESs and Tec elements are generated following their developmental excision and that some flanking DNA sequences are retained at the circle junctions. In this study we have further analyzed the circle junctions of IESs. Analysis of polymerase chain reaction (PCR) products derived from IES circle junctions indicates that at least two sequence arrangements can be present. The circle junctions contain both of the direct repeats that define the ends of the IES separated by either 2 bp flanking the right end of the IES and 8 bp from the left-flanking region, or 8 bp from the right and 2 bp from the left. Using a method that we have termed "strand-biased PCR," we obtained evidence that the junctions of free circular IESs have a 6-base heteroduplex at their center, such that one strand of the DNA is derived from the left-flanking region of the IES and the other from the right. Models of IES excision are presented that incorporate these results and those of previous studies on the excision process.

Developmentally controlled genomic rearrangements in ciliated protozoa.

The ciliated protozoa undergo an extensive genome reorganization during the course of forming a transcriptionally active macronucleus. The process includes numerous chromosome fragmentation and DNA breakage and rejoining events. Recent work indicates that transposable elements play a role in the process.

Sequence of a Euplotes crassus macronuclear DNA molecule encoding a protein with homology to a rat form-I phosphoinositide-specific phospholipase C.

A 604-base pair macronuclear DNA molecule from the hypotrichous ciliate Euplotes crassus was cloned and its DNA sequence determined. The DNA sequence contains an open reading frame capable of encoding a protein 141 amino acids in length. The putative protein contains significant sequence similarity to other eukaryotic proteins, including the rat form-I phosphoinositide-specific phospholipase-C.

High fidelity developmental excision of Tec1 transposons and internal eliminated sequences in Euplotes crassus.

Following the sexual phase of its life cycle, the hypotrichous ciliate Euplotes crassus transforms a copy of its chromosomal micronucleus into a transcriptionally active macronucleus containing short, linear, gene-sized DNA molecules. Tens of thousands of DNA breakage and joining, or splicing, events occur during macronuclear development. The DNA removed by such events includes transposon-like elements, referred to as Tec1 elements, as well as segments of unique sequence DNA, termed internal eliminated sequences (IESs). Both types of elements are bounded by short direct repeats. In the current study, a polymerase chain reaction (PCR) and DNA sequencing strategy has been used to examine the fidelity of excision of two Tec1 elements and three IESs. In all cases, the vast majority of excision events were found to be precise, with one copy of the terminal direct repeats retained at the empty site in the macronuclear DNA molecule. These results, in combination with previous studies that have characterized the excised DNA elements, indicate that the two products of excision (the free element and the macronuclear DNA molecule) share DNA sequences. This suggests that excision events are initiated by staggered cuts in the chromosomal DNA.

Differential DNA amplification and copy number control in the hypotrichous ciliate Euplotes crassus.

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.

Internal eliminated sequences are removed prior to chromosome fragmentation during development in Euplotes crassus.

The hypotrichous ciliated protozoa undergo a massive genome rearrangement process after their sexual cycle. One frequent type of rearrangement is the removal of DNA sequences (internal eliminated sequences; IESs) from internal regions of DNA molecules. In this study, we characterized the removal of IESs in Euplotes crassus. Southern hybridization analyses combined with cytological observations indicated that IES removal is an early event in macronuclear development, occurring during the polytene chromosome stage and prior to the chromosome fragmentation process. The results are consistent with IES removal occurring via an intramolecular DNA breakage and rejoining process.