Hormonal regulation of H19 gene expression in prostate epithelial cells.

The H19 gene is transcribed in an mRNA-like noncoding RNA. When tumors of various organs or cell types are considered, H19 oncogene or tumor-suppressor status remains controversial. To address the potential regulation of H19 gene expression by an androgen steroid hormone (DHT: dihydrotestosterone) or by a peptidic hormone (PRL: prolactin), we performed experiments in rats systemically treated with chemical mediators. This range of in vivo experiments demonstrated that chronic hyperprolactinemia upregulated the H19 expression in epithelial and stromal cells whereas DHT downregulated the gene. PRL and DHT appeared to be opposite mediators in the H19 RNA synthesis. We investigated these hormonal effects in three human prostate epithelial cell lines. In LNCaP cancer cells, the opposite effect of PRL and DHT was corroborated. However, in normal cells (PNT1A), H19 remained insensitive to the hormones in fetal calf serum (FCS) medium but became responsive in a serum-stripped medium. In the DU-145 cancer cell line, tested for its androgen-independence and aggressiveness, the hormones had no effect on H19 expression whatever the culture conditions. Finally, we demonstrated that PRL upregulated the H19 expression in LNCaP cells by the JAK2-STAT5 transduction pathway. We conclude that H19 expression is regulated by both a peptidic and a male steroid hormone.

Steroid hormones modulate H19 gene expression in both mammary gland and uterus.

H19 is an imprinted and developmentally regulated gene whose product remains apparently untranslated. In a previous study on breast adenocarcinomas, we reported that overexpression of the H19 gene was significantly correlated with the presence of steroid receptors, suggesting the putative role of hormones in H19 transcription. To determine the mode of steroid action, we have detected levels of H19 RNA synthesis during mammary gland development by in situ hybridization (ISH): two peaks of H19 transcription occur during puberty and pregnancy. Furthermore, we demonstrated by ISH that in the uterus H19 RNA synthesis is high during estrus and metestrus phases. To test steroid control of H19 transcription, ovariectomized and adrenalectomized mice were supplemented, 1 week after surgery, with 17-beta-estradiol (E2, 20 microg/kg/day), progesterone (P, 1 mg/kg/day) or corticosterone (B, 0.3 mg/ kg/day) for 2 weeks. According to ISH data, E2 and to a lesser extent B stimulated H19 transcription in the uterus, whereas P inhibited it. To confirm the in vivo results, in vitro experiments were performed using cultures of MCF-7 cells (a hormone-sensitive mammary cell line). E2 stimulated the endogenous H19 gene of this cell line and tamoxifen inhibited this effect. Furthermore, we performed transient cotransfections in MCF-7, in HBL-100 (another hormone-sensitive mammary cell line) and in BT-20 (a hormone-insensitive mammary cell line) with various constructs of ERalpha (WT or mutated) and PR-A, in presence or absence of steroid hormones. We demonstrated that ERalpha up-regulated the H19 promoter in MCF-7 and in HBL-100, whereas PR-A did not have any effect per se. Moreover, in MCF-7, PR-A antagonized clearly the ERalpha-mediated promoter enhancement, but in HBL-100 this counteracting effect on the ERalpha up-regulation was not found. Interestingly, the same experiments performed in BT-20 cell line provided very similar results as those obtained in MCF-7 cells, with a clear down-regulation mediated by PR-A on the H19 promoter. All these in vitro data are in agreement with in vivo results. In addition, data obtained with ERalpha mutants indicate that H19 promoter activation is both ligand-dependent and ligand-independent. We have thus demonstrated that H19 gene expression is controlled by steroid hormones; furthermore, this gene is highly expressed in hormone-sensitive organs when the hormonal stimulation is accompanied with a morphological repair.

H19 overexpression in breast adenocarcinoma stromal cells is associated with tumor values and steroid receptor status but independent of p53 and Ki-67 expression.

In a previous study we described the expression of the H19 gene by in situ hybridization (ISH) in normal breast and in benign or malignant breast tumors (Dugimont T, Curgy JJ, Wernert N, Delobelle A, Raes MB, Joubel A, Stehelin D, Coll J: Biol Cell 1995, 85:117-124). In the present work, 1) we extend the previous one to a statistically useful number of adenocarcinomas, including 10 subclasses, 2) we provide information on the precise ISH localization of the H19 RNA by using, on serial tissue sections, antibodies delineating specifically the stromal or the epithelial component of the breast, and 3) we consider relationships between the H19 gene expression and various clinicopathological information as tumor values (T0 to T4), grades, steroid receptors, lymph node status, and molecular features as the p53 gene product and the Ki-67/MIB1 protein, which is specific to proliferating cells. Data indicate that 1) in 72.5% of studied breast adenocarcinomas an overall H19 gene expression is increased when compared with healthy tissues, 2) the H19 gene is generally overexpressed in stromal cells (92.2%) and rarely in epithelial cells (2.9% only), 3) an up-regulation of the H19 gene is significantly correlated with the tumor values and the presence of both estrogen and progesterone receptors, and 4) at the cellular level, the H19 gene demonstrates an independent expression versus accumulation of both the p53 protein and the Ki-67/MIB-1 cell-cycle marker.

The H19 TATA-less promoter is efficiently repressed by wild-type tumor suppressor gene product p53.

The developmentally regulated H19 gene displays several remarkable properties: expression of an apparently non-translated mRNA, genomic imprinting (maternal allele only expressed), relaxation of the imprinting and/or epigenetic lesions demonstrated in some tumors. Despite several observations after relaxation of imprinting status of the gene, data on trans and cis-acting factors required for the human H19 gene expression are still missing. As a first approach to address identification of factors involved in the regulation of the gene, we found that cells from a p53 antisense-transfected HeLa clone displayed increased amounts of H19 transcripts when compared to the non-transfected cells. Moreover, a HeLa clone stably transfected with a temperature sensitive (ts) 143 Ala p53 mutant exhibited temperature-dependent regulation of H19 expression. This preliminary indication of the repressing effect of the p53 protein on H19 expression has been confirmed by transient cotransfection experiments in HeLa cells, using luciferase surrogate constructs under the control of the 823 bp sequence immediately upstream of the transcription start point of the H19 gene, and different constructs containing sense, antisense or a ts 143 Ala mutant p53 cDNA. We observed an increase of H19 promoter-driven activity in transient cotransfections with the antisense p53 cDNA and the temperature sensitive mutant p53 at the non-permissive temperature, but a decrease with sense wild-type p53 cDNA. Furthermore, the cotransfection experiments were repeated in a cell line lacking endogenous p53. (Calu 6 cells) and the results provided additional evidence for a down regulation of the expression of the H19 gene by the p53 protein.

The 5' part of the human H19 RNA contains cis-acting elements hampering its translatability.

H19 is an imprinted gene developmentally regulated in man and mouse and implicated in various neoplasms. No corresponding protein product has yet been detected, although several open reading frames (ORFs) could be identified along its RNA. The largest ORF found in the human gene could encode a putative 26 kDa protein. We have isolated two H19 cDNAs (AP and ES) that contain this ORF4 and correspond to incomplete copies of the unique 2.3 kb H19 RNA. In transient expression assays, AP was able to synthesize a 26 kDa protein whereas ES was not. With respect to ORF4, ES exhibits a 536 bp long GC-rich 5' untranslated region, whereas AP contains the last 22 nucleotides of this 5'UTR. Using deletions and point mutations, we have found that the length and probably the secondary structure of the 5'UTR strongly hampers the translatability of the RNA. In addition, a potential role of upstream ORFs (uORFs) was detected as stressed by the enhances translation of a construct mutated in uORF3 overlapping ORF4. Interactions between H19 and proteins are indicated by a specific binding between 5'UTR derived RNA segments and two nuclear proteins of about 27 kDa. Our results favor a potential role of these particular structures and binding properties in general trans-regulation of RNA post-transcriptional processes rather than in normal control of H19 mRNA translation.

The H19 gene is expressed within both epithelial and stromal components of human invasive adenocarcinomas.

In a previous work, we have isolated the human H19 gene and shown accumulation of transcripts in various human tumors including breast carcinomas (Douc-Rasy et al (1993) Int J Oncol 2, 753-758). Questions arose, after Northern blot results, about the precise H19 mRNA location, specially in normal breast tissues and benign or malign primary breast tumors. Then we performed molecular in situ hybridization to get insight into tissue expression of the H19 gene. Examined resections included one normal tissue, one fibroadenoma and 13 cancers. Results obtained with the H19 probe can be summarized as follows: 1) in normal breast tissues signals were focally observed in epithelial cells, but more predominantly in the palleal tissue which is sensitive to hormones; 2) in the fibroadenoma, fibroblastic cells were extensively labeled at the stroma-epithelium boundary, but epithelial cells were negative; and 3) in primary cancers, eight specimens exhibited signals on stromal cells, one specimen on epithelial cells and four on both epithelial and stromal cells. Data provide the following evidence: 1) usually labeled cells are clustered, either within normal or pathological tissues; 2) the labeling pattern highly differs from one tumor to another; and 3) H19 probe displays very different signals from one cell to another in given compartment of a given tissue section. In conclusion, it seems that a high H19 expression matches the tumor invasion. Our results suggest that the expression of this gene is concerned by the relationships between epithelial and stromal cells, and can reflect peculiar physiological states of the cells. Furthermore, we discuss results showing an abundant expression of H19 gene in some adenocarcinomas of bad prognosis, in the context of the otherwise established tumor-suppressor role of this gene, or the strictly controlled gene dosage, which could be overridden in these particular cases.

Some polypeptides in the nervous system of the marine worm, Nereis diversicolor, are related to the sodium influx stimulating peptide of the pulmonate freshwater snail, Lymnaea stagnalis.

Total mRNA, extracted from brain of the marine worm, Nereis diversicolor (Annelida, Polychaeta), was translated either in vitro using a rabbit reticulocyte lysate or in ovo (Xenopus laevis oocyte). The synthesized polypeptides were analyzed by electrophoresis and Western blotting techniques using polyclonal antisera raised against three peptides: sodium influx stimulating peptide (SISP) sequences 10-19 and 67-76 and a monoclonal antibody raised against purified native SISP (1-77) of Lymnaea stagnalis. Among the products translated in vitro, three polypeptides of 80, 72, and 64 kDa were recognized by the anti-SISP (10-19) polyclonal antiserum and by the monoclonal antiserum, but not by anti-SISP (67-76). Some of the in ovo translated products showed almost identical immunoreactivity to both the anti-SISP (10-19) and the monoclonal antibody. These polypeptides have molecular masses of 80, 72, and 43 kDa. No polypeptides were recognized by anti-SISP (67-76). Western blotting analysis of brain extracts revealed a number of proteins that reacted with the antiserum raised against SISP (10-19) and the monoclonal antiserum. Several perikarya of brain ganglionic nuclei and ventral nerve cord were immunoreactive to anti-SISP (10-19). The monoclonal antiserum gave similar results, although with a less intense immunoreaction. The infracerebral region was also stained, suggesting that the immunoreactive material is released as a true neurohormone into the hemolymph. The largest polypeptides, in particular those translated from brain mRNA, could be neuropeptide precursors containing a SISP-related sequence.

Precursors of molecules related to mammalian opioid peptides in brain of a marine worm.

Total mRNA were extracted from brain of Nereis diversicolor (Annelida, Polychaeta) and were translated in vitro or in ovo. The newly synthesized polypeptides were analyzed through electrophoresis of immunoprecipitated products or the Western blotting technique using polyclonal antibodies raised against mammalian dynorphin 1-17 and mammalian alpha-neo-endorphin. Among the products translated in vitro, only one class of polypeptide of 70 kDa was recognized by anti-dynorphin 1-17 antibodies. Furthermore, some in ovo translated products as well as proteins extracted from brain of worms showed identical immunoreactivity. These polypeptides, 60-70 kDa, reacted with anti-dynorphin 1-17 and anti alpha-neo-endorphin antibodies. These results suggest the existence of epitopes common to in ovo and in vitro translated products, to polypeptides extracted from the brain and to some mammalian opioid peptides of the prodynorphin family. We postulate the presence, in the brain of N. diversicolor, of precursors of peptides related to mammalian dynorphin 1-17 and alpha-neo-endorphin. Data reported in this investigation do not allow us to propose or even postulate the presence, in the brain of the worm, of one precursor molecule common to polypeptides related to mammalian dynorphin 1-17 and alpha-neo-endorphin. Furthermore, the Nereis precursor molecules exhibit a clear-cut difference in molecular mass with the mammalian prodynorphin: 70 kDa versus 30 kDa.

Precursors of neuropeptides in the marine worm Nereis diversicolor. In vitro translation of a precursor related to human prepro-cholecystokinin and immunolocalization of this precursor in the nervous system.

Total mRNA extracted from the brain of a marine worm, Nereis diversicolor, were in vitro translated using 2 cell-free systems: rabbit reticulocyte lysate and wheat germ extract. Among numerous products newly translated in both systems, only one class of 70 kDa polypeptides immunoprecipitated when we used a mixture of 3 well defined antibodies raised against known sequences of the human prepro-CCK. At the cellular level, using immunocytochemistry techniques, strong and moderate immunoreactivities were seen in perikarya located in various ganglionic nuclei of the worm brain. Immunoreactive nerve fibres were visible in the neuropile but not in the infracerebral region, a neurohemal area. Immunoreactions also appeared on perikarya located in the anterior and medial groups of the ventral nerve cord. Furthermore, immunolabeled cells were observed in the midgut. Interestingly, several co-localizations of materials immunologically related to human prepro-CCK and CCK/gastrin were observed in the brain and the ventral nerve cord both in perikarya and in nerve fibres. We propose that, in Nereis a polypeptide (molecular mass 70 kDa) is the large precursor of molecules related to those of the CCK/gastrin peptide family.

Polypeptides related to mammalian procholecystokinin in the brain of an invertebrate, a marine worm, Nereis diversicolor: evidence from in ovo translation of mRNA.

Total mRNA extracted from brain of a sea worm Nereis diversicolor (Annelida, Polychaeta) were injected into Xenopus oocytes. The in ovo-translated polypeptides were analyzed through electrophoresis of immunoprecipitated products or Western blotting techniques. Some of these polypeptides cross-reacted antibodies elaborated against three mammalian (human and rat) procholecystokinin (pro-CCK) sequences. Polypeptides of 15, 64, and 70 kDa were determined, but two families of less obvious products, whose molecular masses were between 27-34 kDa and 46-60 kDa, appeared. Furthermore, the Western blotting technique also showed a cross-reaction between some of the polypeptides (64 and 70 kDa) extracted from brains and the antibodies used. From this work, evidence is given of the presence of epitopes shared by cellular polypeptides extracted from the brain, in ovo-translated products and mammalian pro-CCK. Furthermore, these data suggest the existence of a CCK precursor in the brain of N. diversicolor.

Expression of calcium channels in Xenopus oocyte injected with crab skeletal muscle fibre mRNA.

Using the whole cell voltage-clamp technique and a C1 free and Na free Ba methane sulfonate solution, stage V and VI Xenopus oocytes demonstrated a Ba current (endogenous component) with a peak amplitude average of 6 nA (6 +/- 2 nA). When oocytes were injected with crustacean skeletal muscle mRNA, an additional component of IBa could be detected (exogenous IBa). The latter current could be distinguished from the native one by several electrophysiological means: a peak amplitude average of 90 nA (90 +/- 4 nA), activation potential threshold, steady state inactivation properties and sensitivity to Ca blockers. As shown by Jdaïâa and Guilbault in crustacean skeletal muscle fibres, exogenous IBa could be divided into two components: a "fast component" and a "slow component" probably passing through two types of Ca channels (fast and slow) since the peak Ba current voltage relationship was biphasic and the fast component of exogenous IBa was less sensitive than the slow to nifedipine. The features of the newly synthesized channels incorporated in the Xenopus oocyte membrane suggest that they may be associated with fast and slow channels, previously described in many preparations, particularly in crustacean skeletal muscle fibres.

Translation in a cell-free system, and in Xenopus oocytes, of mRNA which specify a cadmium-binding protein in Nereis diversicolor (Annelida, Polychaeta).

The presence in the marine worm Nereis diversicolor of a low molecular mass protein with the capacity to bind cadmium has been previously demonstrated. Poly(A)(+)-mRNA were extracted from coelomocytes of Nereis diversicolor and were translated either in vitro, using a rabbit reticulocyte lysate, or in vivo into Xenopus laevis oocytes. Analysis of synthesized polypeptides by enzyme-linked immunosorbent assay (ELISA) and by Western blotting, using a specific monoclonal anti-MP II antibody, showed that this metalloprotein was translated both in in vitro and in vivo translation systems, with an apparent molecular mass of 11-13 kDa. Two other products, with 26.5 and 28 kDa molecular mass, cross-reacted with the monoclonal anti-MP II antibodies. The present work confirms that coelomocytes are sites of important synthesis of MP II-mRNA.

Subunit arrangement of Escherichia coli F1-ATPase studied by scanning transmission electron microscopy.

The shape and the arrangement of subunits in Escherichia coli F1-ATPase (ECF1) lacking the delta subunit have been explored with a high performance scanning transmission electron microscope. In tilting experiments, the ECF1 molecule appeared as a flat cylinder whose width (approx. 120 A) was about twice its height. The symmetry of front view projections of ECF1 has been investigated by computer analysis. In a population taken at random from the data bank, one third of the particles showed five-fold radial symmetry components, one third six-fold radial symmetry components and the last third no typical symmetry. The six-fold radial symmetry was consistent with a hexagonal arrangement of six large peripheric masses, which probably correspond to the three alpha and the three beta subunits of ECF1. The five-fold radial symmetry was tentatively explained by a fusion of two juxtaposed peripheric subunits. Lateral projections showed a zig-zag organization of the large masses, suggesting that the large alpha and beta subunits are located on two levels, with some degree of intercalation between the subunits of the two levels.

The mitoribosomes.

So great an interest in numerous laboratories toward the understanding of the mitoribosome structures and functions comes from at least the two following considerations: a) in the field of the biogenesis of mitochondria, the mitoribosome is a biological key structure, on which synthesis of fundamental mitochondrial elements depends; b) from a phylogenetic point of view questions about the ancestral origin of mitochondrial genomes remain open. Mitoribosomes resemble other ribosomes in their fundamental properties. They are constituted of two subunits containing RNA and proteins. They function according to the same overall mechanism, using initiator tRNA, aminoacyl-tRNA and factors for initiation and elongation to translate mRNA. Mitoribosomes have been observed in situ and their composition has been established by ultrastructural cytochemistry. They are preferentially associated with the inner mitochondrial membrane and are occasionally aligned in "mitopolysomes". Mitoribosomes have been isolated, obtained whole or dissociated into subunits. Frequently, fine morphological details permit to distinguish the mitoribosomes from their cytoplasmic counterparts. The diversity found in various physico-chemical properties (S coefficient, molecular weight, buoyant density, RNA/protein ratio, RNA and protein characteristics) of mitoribosomes indicates that this class of ribosomes is the more heterogeneous. Small and large mrRNA from various organisms showed frequent homologies and conserved basic secondary structures (these similarities depending on the organism) in defined RNA regions, when compared with their counterpart molecules in other ribosomes from various origins. These regions are probably involved in the maintenance of fundamental active conformation. Post-transcriptional oligoadenylation of 3'-termini of the small and large mrRNA in mammals appears to be a general phenomenon. Methylated nucleotides in large and small mrRNA are rare, but their presence seems to constitute an important feature, for they have been phylogenetically conserved and are located in regions of the mrRNA molecules which show a high degree of primary sequence conservation. One unique feature of the mitoribosomes of animal and fungal cells is the absence of 5 S and 5.8 S rRNA molecules. But a well-established exception to the general absence of 5 S RNA is the presence of this RNA molecule in mitoribosomes extracted from higher plants.(ABSTRACT TRUNCATED AT 400 WORDS)

A STEM approach to the study of the proteinaceous architecture of a ribosome.

We used a high-resolution STEM to attempt to recognize the spatial localization of the macromolecular components of ribosomes isolated from Tetrahymena pyriformis. Images obtained either by the annular dark field detector or by the ratio-contrast mode (Z-contrast) show a ribosome as constituted of rounded dark areas, the number and size of which are compatible with those of the protein molecules recognized by biochemistry. Images of different ribosomes, when similar orientations are observed, exhibit a roughly constant pattern of the dark areas. A set of images of a tilted ribosome allows to recognize that some masses are in fact two more superimposed components. We propose that the dark areas represent essentially the proteinaceous architecture of the ribosome.

Effects of chloramphenicol on the mitochondrial respiratory chain in the wild strain and in a cytoplasmic chloramphenicol-resistant mutant of Tetrahymena pyriformis.

The effects of chloramphenicol (CAP) on mitochondrial respiratory activity in the wild strain (ST) and in a cytoplasmic CAP-resistant mutant (STR1) of Tetrahymena pyriformis were studied by determining oxygen consumption, by spectrophotometry, and by cytochemistry. In the absence of CAP both strains had the same respiration capacity, and the low-temperature spectra of their isolated mitochondria were similar. Furthermore, the mitochondria of both strains showed a positive reaction with diaminobenzidine, denoting a similar cytochrome oxidase activity. However, when cells were grown in CAP for 24 or 48 h, the peaks of cytochrome oxidase and cytochromb b were almost absent in the wild type. In this type the oxygen consumption was greatly decreased, and the mitochondria were no longer stained by diaminobenzidine. In the mutant, the peaks of cytochrome oxidase and cytochrome b were decreased only; respiration was less affected than in the wild type, and cytochrome oxidase activity was still disclosed by the diaminobenzidine reaction. These results show that CAP inhibits the synthesis of two cytochromes (b and oxidase) which are partially translated into the mitochrondria of T. pyriformis. In the mutant, CAP reduces only the mitochondrial translation, resulting in reduced mitochondrial activity and reduced growth rate of the cell. These results are compared with the nucleo-mitochondrial regulation mechanisms discussed in our previous works.

The mitoribosomes of a chloramphenicol-resistant cytoplasmic mutant of Tetrahymnea pyriformis differ from those of the wild strain.

The spontaneous CAP-resistant mutant, STR1, has been isolated from the sensitive St-strain of Tetrahymena pyriformis (Curgy et al., Biologie Cellulaire 37, 51-60, 1980; Perasso et al., Biologie Cellulaire 37, 45-50, 1980). The goal of the present work is to disclose if the resistance character is due to a modification in the mitoribosomes and if the CAP-treatment induces changes in their abundance and in their physico-chemical properties.The results show that the resistance character of the mutant is due to a reduced affinity of its mitoribosomes for CAP. This difference can be explained by modifications of at least one protein which is probably coded for by the mitochondrial genome.The mitoribosomes from CAP-treated sensitive cells tend to dissociate into their subunits and the electrophoretic pattern of their proteins suggests that at least two mitoribosomal proteins are necessary to bound the two subunits together. These proteins are probably translated in mitochondria.Finally, the CAP-treatment induces a decrease of the abundance of mitoribosomes in the sensitive cells whereas it induced an increase in the resistant cells. The latter change can be regarded as a regulatory mechanism owing to which a loss of efficiency of the mitoribosomes is compensated by their enlarged abundance.

[Analysis of ribosomes by polyacrylamide gel electrophoresis (author's transl)]. / Analyse de ribosomes par électrophorèse en gel de polyacrylamide

Ribosomal polymers, monomers and subunits from several eukaryotes and prokaryotes were isolated and analyzed by polyacrylamide gel electrophoresis. Extraction of RNA from ribosomal particles after their migration in a polyacrylamide gel, analyses by sedimentation in sucrose gradients and observations in the electron microscope were carried out in parallel. Attention was directed to the reproducibility, the precision and the limitations of the electrophoresis technique.

Mitochondrial and cytoplasmic ribosomes from Tetrahymena pyriformis. Correlative analysis by gel electrophoresis and electron microscopy.

Mitochondrial and cytoplasmic ribosomes from Tetrahymena pyriformis have been isolated and studied by the techniques of polyacrylamide gel electrophoresis and electron microscopy used in conjunction. Although the two ribosome types show the same coefficient of sedimentation (80S) in sucrose gradients, they can be distinguished by gel electrophoresis: mitoribosomes migrate in a single band, considerably slower than the cytoribosome band. Electron microscope observations of negatively stained cytoribosomes show typical rounded or triangular profiles, about 275 x 230 A; mitoribosome profiles are much larger and clearly elongate, about 370 x 240 A. An electron-opaque spot delimits two nearly equal size subunits. In mixtures of mito- and cytoribosomes, each type can be recognized by its characteristic electrophoretic mobility and by its distinctive fine structure. Cytoribosomal 60S and 40S subunits each produce a distinct electrophoretic band. On the contrary, neither electrophoretic analysis, using a variety of conditions, nor electron microscopy is able to discern two different subunit types in the single 55S mitoribosomal subunit peak. Electrophoretic analysis of RNA shows that both ribosomal RNA species are present in the mitoribosomal subunit fraction. These results establish that mitoribosomes from T. pyriformis dissociate into two subunits endowed with the same sedimentation coefficient, the same electrophoretic mobility, and a similar morphology.