Project description:BACKGROUND: There are thousands of very diverse ciliate species from which only a handful mitochondrial genomes have been studied so far. These genomes are rather similar because the ciliates analysed (Tetrahymena spp. and Paramecium aurelia) are closely related. Here we study the mitochondrial genomes of the hypotrichous ciliates Euplotes minuta and Euplotes crassus. These ciliates are only distantly related to Tetrahymena spp. and Paramecium aurelia, but more closely related to Nyctotherus ovalis, which possesses a hydrogenosomal (mitochondrial) genome. RESULTS: The linear mitochondrial genomes of the hypotrichous ciliates Euplotes minuta and Euplotes crassus were sequenced and compared with the mitochondrial genomes of several Tetrahymena species, Paramecium aurelia and the partially sequenced mitochondrial genome of the anaerobic ciliate Nyctotherus ovalis. This study reports new features such as long 5'gene extensions of several mitochondrial genes, extremely long cox1 and cox2 open reading frames and a large repeat in the middle of the linear mitochondrial genome. The repeat separates the open reading frames into two blocks, each having a single direction of transcription, from the repeat towards the ends of the chromosome. Although the Euplotes mitochondrial gene content is almost identical to that of Paramecium and Tetrahymena, the order of the genes is completely different. In contrast, the 33273 bp (excluding the repeat region) piece of the mitochondrial genome that has been sequenced in both Euplotes species exhibits no difference in gene order. Unexpectedly, many of the mitochondrial genes of E. minuta encoding ribosomal proteins possess N-terminal extensions that are similar to mitochondrial targeting signals. CONCLUSION: The mitochondrial genomes of the hypotrichous ciliates Euplotes minuta and Euplotes crassus are rather different from the previously studied genomes. Many genes are extended in size compared to mitochondrial genes from other sources.
Project description:Programmed translational frameshifts have been identified in genes from a broad range of organisms, but typically only a very few genes in a given organism require a frameshift for expression. In contrast, a recent analysis of gene sequences available in GenBank from ciliates in the genus Euplotes indicated that >5% required one or more +1 translational frameshifts to produce their predicted protein products. However, this sample of genes was nonrandom, biased, and derived from multiple Euplotes species. To test whether there truly is an abundance of frameshift genes in Euplotes, and to more accurately assess their frequency, we sequenced a random sample of 25 cloned genes/macronuclear DNA molecules from Euplotes crassus. Three new candidate +1 frameshift genes were identified in the sample that encode a membrane occupation and recognition nexus (MORN) repeat protein, a C(2)H(2)-type zinc finger protein, and a Ser/Thr protein kinase. Reverse transcription-PCR analyses indicate that all three genes are expressed in vegetatively proliferating cells and that the mRNAs retain the requirement of a frameshift. Although the sample of sequenced genes is relatively small, the results indicate that the frequency of genes requiring frameshifts in E. crassus is between 3.7% and 31.7% (at a 95% confidence interval). The current and past data also indicate that frameshift sites are found predominantly in genes that likely encode nonabundant proteins in the cell.
Project description:We have identified two 1.6 kb macronuclear DNA molecules from Euplotes crassus that hybridize to the alpha subunit of the Oxytricha telomere protein. We have shown that one of these molecules encodes the 51 kDa Euplotes telomere protein while the other appears to encode a homolog of the telomere protein. Although this homolog clearly differs in sequence from the Euplotes telomere protein, the two proteins share extensive amino acid sequence identity with each other and with the alpha subunit of the Oxytricha telomere protein. In all three proteins 35-36% of the amino acids are identical, while 54-56% are similar. The most extended regions of sequence conservation map within the N-terminal section; this section has been shown to comprise the DNA-binding domain in the Euplotes telomere protein. Our findings suggest that some of the conserved amino acids may be involved in DNA recognition and binding. The gene encoding the telomere protein homolog contains two introns; one of these introns is only 24 bp in length. This is the smallest mRNA intron reported to date.
Project description:More than 100,000 interstitial segments of DNA (internal eliminated sequences [IESs]) are excised from the genome during the formation of a new macronucleus in Euplotes crassus. IESs include unique sequence DNA as well as two related families of transposable elements, Tec1 and Tec2. Here we describe a new class of E. crassus transposons, Tec3, which is present in 20 to 30 copies in the micronuclear genome. Tec3 elements have long inverted terminal repeats and contain a degenerate open reading frame encoding a tyrosine-type recombinase. One characterized copy of Tec3 (Tec3-1) is 4.48 kbp long, has 1.23-kbp inverted terminal repeats, and resides within the micronuclear copy of the ribosomal protein L29 gene (RPL29). The 23 bp at the extreme ends of this element are very similar to those in other E. crassus IESs and, like these other IESs, Tec3-1 is excised during the polytene chromosome stage of macronuclear development to generate a free circular form with an unusual junction structure. In contrast, a second cloned element, Tec3-2, is quite similar to Tec3-1 but lacks the terminal 258 bp of the inverted repeats, so that its ends do not resemble the other E. crassus IES termini. The Tec3-2 element appears to reside in a large segment of the micronuclear genome that is subject to developmental elimination. Models for the origins of these two types of Tec3 elements are presented, along with a discussion of how some members of this new transposon family may have come to be excised by the same machinery that removes other E. crassus IESs.
Project description: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.
Project description:The deposisted data is the deep dive global shotgun bottom-up LC-MS/MS proteome analysis of the Euplotes crassus microorganism. The main objective of the project was to find confirmation of the frameshifts at the protein level.
Project description:The macronuclear genome of Euplotes crassus contains two different genes, EFA1 and EFA2, encoding EF-1alpha proteins. They are derived from micronuclear precursors in the course of a sexual process termed conjugation. We have found that two apparent micronuclear precursors exist for EFA1. They differ in their potential coding sequences and in the internal sequences interrupting the genes, which are normally removed during the processing from micronuclear into macronuclear genes. One of these genes is not processed into a macronuclear gene and has accumulated C-->T transitions in a limited section of the coding region. The gene obviously constitutes a recent duplication which has lost its ability to be processed into a macronuclear gene and has therefore become a micronuclear pseudogene. The true EFA1 precursor harbours a novel type of internal sequence in addition to a classical AT-rich IES. As usual, only one micronuclear EFA2 precursor gene was found. Its coding sequence is interrupted by a 79 bp TeIIES.
Project description:Telomere end binding proteins from diverse organisms use various forms of an ancient protein structure to recognize and bind with single-strand DNA found at the ends of telomeres. To further understand the biochemistry and evolution of these proteins, we have characterized the DNA binding properties of the telomere end binding protein from Euplotes crassus (EcTEBP). EcTEBP and its predicted amino-terminal DNA-binding domain, EcTEBP-N, were expressed in Escherichia coli and purified. Each protein formed stoichiometric (1:1) complexes with single-strand DNA oligos derived from the precisely defined d(TTTTGGGGTTTTGG) sequence found at DNA termini in Euplotes. Dissociation constants for DNA x EcTEBP and DNA x EcTEBP-N complexes were comparable: K(D-DNA) = 38 +/- 2 nM for the full-length protein and K(D-DNA) = 60 +/- 4 nM for the N-terminal domain, indicating that the N-terminal domain retains a high affinity for DNA even in the absence of potentially stabilizing moieties located in the C-terminal domain. Rate constants for DNA association and DNA dissociation corroborated a slightly improved DNA binding performance for the full-length protein (ka = 45 +/- 4 microM(-1) s(-1), kd = 0.10 +/- 0.02 s(-1)) relative to that of the N-terminal domain (ka = 18 +/- 1 microM(-1) s(-1), kd = 0.15 +/- 0.01 s(-1)). Equilibrium dissociation constants measured for sequence permutations of the telomere repeat spanned the range of 55-1400 nM, with EcTEBP and EcTEBP-N binding most tightly to d(TTGGGGTTTTGG), the sequence corresponding to that of mature DNA termini. Additionally, competition experiments showed that EcTEBP recognizes and binds the telomere-derived 14-nucleotide DNA in preference to shorter 5'-truncation variants. Compared with the results for multisubunit complexes assembled with telomere single-strand DNA from Oxytricha nova, our results highlight the relative simplicity of the E. crassus system where a telomere end binding protein has biochemical properties indicating one protein subunit caps the single-strand DNA.
Project description: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.
Project description:The analysis of a repetitive DNA interruption of the micronuclear precursor to a 0.85-kb macronuclear gene in the hypotrich Euplotes crassus has led to the identification of a second transposon-like element named Tec2. Two copies of this element, one inserted into the other, compose the interruption. The Tec2 element resembles the previously characterized Tec1 element in overall size, copy number, length, and extreme terminal sequence of its inverted repeats and in the apparent use of a 5'-TA-3' target site. In addition, extrachromosomal circular forms of Tec2 appear in DNA isolated from cells undergoing macronuclear development at the same time and with the same conformation as extrachromosomal circular forms of Tec1. These similarities suggest that the Tec1 and Tec2 elements may be under the same type of regulation during macronuclear development.