The pheromone 3-encoding gene (phr3) of Euplotes octocarinatus was expressed in Escherichia coli using a novel expression-secretion vector. The vector, pExSec1, contains a strong and tightly ...regulated T7 promoter, the corresponding Shine-Dalgarno sequence and the T7 terminator region. Translation starts at the protein A leader sequence followed by the synthetic ZZ sequence of protein A. The expression-secretion modules are embedded in the high-copy-number plasmid vector, pUK21, which carries a kanamycin-resistance marker (KmR). The produced ZZ-pheromone 3 (Phr3) fusion protein was secreted into the culture medium of the host cells. It was isolated by affinity chromatography and was further purified by gel filtration. After refolding with protein disulfide isomerase (PDI), the fusion protein exhibited the same high activity as the native pheromone.
Several polyclonal antibodies raised against distinct γ-tubulin sequences previously showed the presence of γ-tubulin in the cortical MTOCs (basal bodies of dorsal cilia, cirri and membranelles) and ...also in the micronucleus 21. In using one of these antibodies during the successive stages of conjugation, we analysed by immunofluorescence the precise dynamics of the localization of γ-tubulin in the intranuclear mitotic and meiotic nuclei, whose spindle, like in other ciliates, is assembled without visible polar structures. The interphase micronucleus exhibits a spotted distribution of γ-tubulin. In prophase, the labeling is weaker than in interphase, but remains punctate. When the chromatin condense during metaphase, the γ-tubulin exhibits a longitudinal distribution similar to the chromatin repartition. At the end of metaphase and beginning of anaphase, this labeling condenses into an equatorial region, between the two migrating clusters of chromosomes. This equatorial labeling becomes thinner until it disappears in late anaphase A. At this time, both extremities of the spindle are strongly labeled. Finally, in telophase the two daughter nuclei already show the spotted interphase labeling, while a relocalization of γ-tubulin results in a strong decoration at the level of the intermediate portion of the long spindle that separates the two nuclei. We also report the detection of γ-tubulin in the macronuclear anlage only during certain stages of its development, and raise the question of its significance. Our observations on Euplotes, although not entirely identical to those made on several evolutionarily distant organisms (for review see 13), demonstrate that the relocalization of γ-tubulin during the successive mitotic stages are of general occurrence.
A polypeptide, termed gamone 1, was isolated and purified to homogeneity from culture filtrates of mating type VII of the freshwater ciliate
Euplotes octocarinatus. The gamone induces intraclonal ...conjugation in cells of certain other mating types. The isolation and purification of the gamone was carried out by a combination of two chromatographic steps. The purified gamone was found to be still effective in a concentration of approx. 10
−15M.
The alpha- and the beta-tubulin genes of the hypotrichous ciliate Euplotes octocarinatus were isolated from a size-selected macronuclear DNA library. The alpha-tubulin gene is located on a 1,587 bp ...macronuclear DNA molecule and the beta-tubulin gene on a 1,524 bp macronuclear DNA molecule. Sequencing revealed that all the cysteine residues of the two genes are encoded by the common cysteine codons UGU and UGC and none by an UGA codon. This is in contrast to the genes of E. octocarinatus sequenced so far, where some of the cysteines are encoded by the opal codon UGA. The tubulin genes end like other Euplotes genes with a TAA. They do not contain introns. The last codon for an amino acid in the alpha-tubulin gene is a GAA which codes for glutamic acid. This is in contrast to what has been reported for most alpha-tubulin genes, but it supports for other hypotrichous ciliates. No evidence for the existence of more than one type of alpha- and one type of beta-tubulin genes could be obtained.
The genes encoding pheromones 1 and 2 of
Euplotes octocarinatus are located on 1761 bp and 1763 bp long macronuclear DNA molecules. Both molecules as well as the cDNAs corresponding to the genes ...located on these molecules were cloned and sequenced. The genes have the same general structure. In both cases, the sequences of the secreted pheromones are preceded by leader sequences which contain typical ER-signal sequences. Each of the two genes contains three introns, a long one interrupting the leader sequence and two short ones located in the sequence coding for the secreted pheromone. Both genes contain three in-frame TGA stop-codons, presumably encoding cysteines for these pheromones. Very similar or identical non-coding sequences suggest regulatory functions, and putative regulatory consensus sequences are present in these regions.
The data of pheromones 1 and 2, deduced from their genes, complete previous studies on pheromones 3 and 4 of
E. octocarinatus, and allow, by comparison, several general conclusions about the entire pheromone system of this species. Most importantly, all four pheromone genes have a highly similar gene structure with in-frame TGA codons, astonishingly similar or identical non-coding regions (including introns) but rather poor similarities between the secreted pheromone proteins.
