Isolation and characterization of a cDNA encoding a putative high mobility group (HMG) – box protein from stored mRNA in resting cysts of the ciliate Oxytricha (Sterkiella) nova – Ciliate macronuclear gene encoding a putative HMG-box protein

We have isolated an cDNA after applying a DDRT-PCR analysis on mRNA from mature resting cysts of the ciliate Oxytricha (Sterkiella) nova. From this cDNA fragment the complete macronuclear minichromosome was obtained by using the Mac-End-PCR method. After cloning and sequencing, this cDNA shown certain similarity to HMG-like proteins. The analysis of the inferred amino acid sequence shown that this putative HMG-like protein has one HMG-box interrupted by a intron. The analysis of others characteristics (including a 3D model) confirms that it is a HMGB family protein. It is the first time that a macronuclear gene encoding a putative HMG-box protein is isolated from resting cysts of a stichotrich ciliate. The possible implications of this stored mRNA in the ciliate cryptobiotic stage are discussed.     

A structurally deviant member of the Euplotes raikovi pheromone family: Er-23

Pheromones of Euplotes raikovi form a homologous family of proteins with 37- to 40-amino acid residues, including six cysteines that form three strictly conserved disulfide bridges. The determination of the primary structure of the pheromone Er-23, which was isolated from cells derived from natural populations of E. raikovi that secrete the other known pheromones, has now revealed a novel structure type. The polypeptide chain of this pheromone contains 51 residues, 10 of which are cysteines presumably involved in the formation of five disulfide bridges, and lacks a carboxyl-terminal tail following the last cysteine of the sequence. The elongation of the Er-23 molecule is presumed to result from multiple events of gene duplication starting from an ancestral motif Xxx(2-4)-Cys-Xxx(5-7)-Cys.     

Analysis of mating type determination by transplantation of O macronuclear karyoplasm in Paramecium tetraurelia

The odd (O) or even (E) mating type in Paramecium tetraurelia is determined during the first cell cycle after new macronuclear development. The present paper demonstrates that mating type E is irreversibly determined at the end of the first cell cycle. Direct evidence comes from transplanting O macronuclear karyoplasm containing O-determining factor into E autogamous cells during a new postzygotic macronuclear development. Transplantation of O macronuclear karyoplasm into E autogamous cells at 7–8 hr after the origin of the macronucleus from a product of the synkaryon produces nearly 100% O mating type among the exautogamous cell lines but almost none 10–11 hr after the origin of the macronucleus (around the end of the first cell cycle). The macronuclear anlagen at the stage at which mating type E seems to be fixed contains about 20 times as much DNA as the vegetative G1 micronucleus. The O-determining factor shifting E cells toward O mating type by transplanting O macronuclear karyoplasm is also produced by the newly developed macronucleus in an effective concentration at 10–11 hr after the sensitive period and produced at full levels by the third cell cycle. The level of O factor in the macronucleus then gradually declines with subsequent repeated rounds of DNA synthesis and is finally lost by the eighth cell cycle.     

Analysis of the subtelomeric regions of macronuclear gene-sized DNA molecules of the hypotrichous ciliate Stylonychia lemnae: Implications for the DNA fragmentation process during macronuclear development?

The subtelomeric regions of macronuclear gene-sized DNA molecules from Stylonychia lemnae were analyzed. The results obtained indicate that these regions show a highly ordered and common sequence organization: Immediately adjacent to the telomeric sequence a short inverted repeat sequence is found, followed by another 7–9 bp inverted repeat sequence at approximately position 40. A 10 bp consensus sequence found in the subtelomeric regions of all gene-sized DNA molecules is found at approximately position 60 and in addition at about the same position palindromic sequences showing no homology to each other are localized. The biological significance of this sequence organization is discussed. © 1993Wiley-Liss, Inc.     

Differential Nuclear Coloration in Ciliates

SYNOPSIS. Application of the triple stain of Grosso (methyl green-pyronin-orange G) to the exconjugants of different species of ciliates produces a differential coloration of their nuclei. Both the old macronucleus or its remnants and the micronuclei bind methyl green whereas the newly developing macronucleus (macronuclear anlage) has a selective affinity for the orange G present in the mixture. This finding holds true not only for hypotrichous ciliates, whose macronuclear anlagen are involved in the formation of polytene chromosomes, but also for other ciliates not included in the order Hypotrichida. Such a reaction of the new macronuclei apparently results from the presence of non-histone (acidic) proteins associated with the nucleic acids.
The staining method is fully described to provide a more general application.     

Developmental expression of macronuclear specific antigen in Paramecium caudatum

We obtained a monoclonal antibody (MA-1) specific for macronuclei of the ciliate Paramecium caudotum and P. dubosqui. Immunoblotting showed that the antigen was a poly-peptide of 50 kilodalton (kDa). During the process of nuclear differentiation in P. caudatum, the MA-1 antigens appeared in the macronuclear anlagen immediately after four out of eight post zygotic nuclei differentiated morphologically into the macro-nuclear anlagen. Afterwards, the antigens could be detected in the macronucleus through the cell cycle, and disappeared when the macronucleus began to degenerate in exconjugant cells. These results suggest that the antigens may play a role in the differentiation and function of the macronucleus. © 1992 Wiley-Liss, Inc.     

Tetrahymena Telomerase RNA levels increase during macronuclear development

Telomeres, the G-rich sequences found at the ends of eukaryotic chromosomes, ensure chromosome stability and prevent sequence loss from chromosome ends during DNA replication. During macronuclear development in Tetrahymena, the chromosomes fragment into pieces ranging from 20 kb to 1,500 kb. Tetrahymena telomerase, a ribonucleoprotein, adds telomeric (TTGGGG)_n repeats onto telomeres and onto the newly generated macronuclear DNA ends. We have investigated whether telomerase RNA levels increase during macronuclear development, since such an increase might be expected during chromosomal fragmentation. The steady-state level of the telomerase RNA component was used to estimate the abundance of telomerase present in mating and nonmating Tetrahymena. Northern blot analysis revealed that in vegetatively growing Tetrahymena, there were 18,000–40,000 copies of telomerase RNA per cell. In mating cultures, the levels of RNA increased 2-to 5-fold at 9–15 h, and 1.5- to 3.5-fold in starved nonmating cultures. This increase in telomerase RNA paralleled telomerase activity, which also increased slightly in mating and starved nonmating cells. © 1992 Wiley-Liss, Inc.