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Faithful expression of a heterologous gene carried on an artificial macronuclear chromosome in Euplotes crassus.

ABSTRACT: Macronuclear chromosomes of hypotrichous ciliates are gene-sized molecules carrying the coding sequence flanked by short non-translated regions and bounded by telomeres. We have constructed artificial chromosomes for investigation of transcription in the macronucleus of Euplotes crassus. The neo gene was put under the control of the 5"-non-translated region of the TBP gene of E.crassus. These molecules were introduced into the cell with the help of liposomes. The cells were transformed and survived high concentrations of geneticin. The artificial chromosomes were kept in the macro-nucleus for at least 50 days at a copy number of about 200 per macronucleus. Expression of the gene was shown by reverse transcription of the neo messenger. The transcription start was mapped and found to coincide with that found on the natural macronuclear chromosome encoding TBP in E.crassus.

SUBMITTER: Bender J

PROVIDER: S-EPMC148544 | biostudies-other | 1999 Aug

REPOSITORIES: biostudies-other

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Faithful expression of a heterologous gene carried on an artificial macronuclear chromosome in Euplotes crassus.

Bender J J Kämpfer M M Klein A A

Nucleic acids research 19990801 15

Macronuclear chromosomes of hypotrichous ciliates are gene-sized molecules carrying the coding sequence flanked by short non-translated regions and bounded by telomeres. We have constructed artificial chromosomes for investigation of transcription in the macronucleus of Euplotes crassus. The neo gene was put under the control of the 5"-non-translated region of the TBP gene of E.crassus. These molecules were introduced into the cell with the help of liposomes. The cells were transformed and survi ...[more]

PMID: 10454614

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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:Like many other organisms, ciliates communicate and interact socially via diffusible chemical signals, named pheromones, that are functionally associated with a genetic mating-type mechanism of cell self/not-self recognition. In Euplotes species, pheromones form species-specific families of small, globular, and disulfide-rich proteins folding into exclusively helical secondary structures. Each is specified by one of a series of high-multiple alleles that are inherited in Mendelian fashion with relationships of co-dominance at the so-called mat genetic locus of the cell transcriptionally inert micronuclear genome, and expressed in the transcriptionally active macronuclear genome as individual DNA molecules in which the central coding region is flanked by 5'-leader and 3'-trailer noncoding regions ending with C4A4/T4G4 telomeric repeats. In E. crassus, a cosmopolitan marine species with a long tradition in the study of ciliate mating systems and breeding patterns, oligonucleotides specific to amino acid sequences of pheromones Ec-1 and Ec-α were previously used to clone and sequence a first set of four structurally distinct macronuclear (mac) pheromone coding genes, mac-ec-α, mac-ec-1, mac-ec-2 and mac-ec-3, from two interbreeding strains, L-2D and POR-73. The use of these oligonucleotides in PCR amplifications of macronuclear DNA preparations from three other E. crassus interbreeding strains, ES10, Fava4 and MN4, has now resulted in the characterization of a second set of eight new pheromone coding genes, mac-ec-β, mac-ec-γ, mac-ec-δ, mac-ec-ε, mac-ec-µ, mac-ec-4, mac-ec-5 and mac-ec-6. Multiple alignment between previously and newly determined pheromone-gene sequences reinforces the concept that the E. crassus pheromone-gene family includes two sub-families, which likely reflect a duplication of the micronuclear mat gene locus and represent an apomorphic trait of the E. crassus clade. Members of one sub-family (each identified with a Greek letter) show a 500-bp 5'-leader noncoding region rich in AGGA/AGGGA repetitions, and encode 56-amino acid pheromones with eight conserved Cys residues. Members of the other sub-family (each identified with an Arabic numeral) show an 800-bp 5'-leader noncoding region without AGGA/AGGGA repetitions, and encode 45-amino acid pheromones with ten conserved Cys residues.

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.

Dataset Information

Faithful expression of a heterologous gene carried on an artificial macronuclear chromosome in Euplotes crassus.

Publications

Faithful expression of a heterologous gene carried on an artificial macronuclear chromosome in Euplotes crassus.

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