Quantitative analysis, by ultrastructural in situ hybridization, of mitochondrial genomes and their expression in mid-gut and ovarian cells of a mutant strain of Drosophila subobscura.

In the mitochondrial deletion mutant strain studied here, two types of DNA coexist (heteroplasmy): intact mtDNA (15.9 kb) and mutant mtDNA (10.9 kb), which represents about 80% of the mitochondrial genomes in somatic tissues. The heteroplasmy level is lower in ovary (63%). Mutation is transmitted unchanged through generations. Quantitative analysis of in situ DNA hybridization demonstrated that for the 12SrDNA probe, of a gene outside the deletion, the mitochondrial DNA cellular content in the studied cells of the mutant strain is 1.5 times higher than in the wild-type strain. For the probe encoding Cyto b, a mitochondrial gene affected by the mutation, the ratios (mutant versus wild-type content) differ according to cell type: close to 0.4 in MGE cells and 0.7 in ovary cells. These values indicate heteroplasmic levels of about 72% in MGE cells and 50% in stage 10 oocytes, which is lower than that previously reported for stage 14 oocytes (60%) and embryos (69%). Analysis of in situ RNA hybridization showed that for the 12SrDNA probe, the transcript concentrations do not differ significantly between MGE cells and cells of germinal origin from the two strains. For the Cyto b probe, the mutant RNA/wild-type RNA ratios are lower in somatic cells than in stage 10 nurse cells and oocytes, but in each case less than expected. These studies indicate that the progressive heteroplasmy increase may be related to intense phases of mitochondria biogenesis and that different compensatory phenomena may exist.

Developmental changes in heteroplasmy level and mitochondrial gene expression in a Drosophila subobscura mitochondrial deletion mutant.

Eighty percent of DNA molecules are deleted in the mitochondrial population of an adult mutant strain of D. subobscura. Both intact and deleted genomes are autonomous monomers. The heteroplasmy level, which is lower in germ tissue, increases from the oocytes (60%) to the third larval instar (83%), and is then maintained throughout the life of the fly. The mtDNA/nuclear DNA ratio is on average two-times greater in the heteroplasmic strain than in the wild-type strain, irrespective of the stage, but the cellular content of mitochondria is elevated only in the embryos and pupae of the mutant strain. The steady state concentrations (SSCs) of the transcripts affected by the deletion are greatly reduced at the larval and adult stages, and less so at the pupal stage of the mutant strain compared with the wild-type. The SSCs of these transcripts are identical in the two strains at the embryonic stage. The fusion transcript, indicating that the deleted genome is expressed, was detected at all stages. The mechanisms involved in the changes in the heteroplasmy level during the course of development and in its maintenance from the third larval instar onwards are discussed.

Biochemical and immunological analyses of the flagellin of Clostridium tyrobutyricum ATCC 25755.

The monoclonal antibody 21E7-B12 (IgG3) can be used in a direct method of Clostridium tyrobutyricum detection based on an immunoenzymatic assay. Immunoelectron microscopy demonstrated that the 21E7-B12 antibody recognized the surface-exposed epitopes on the flagellar filaments of C. tyrobutyricum. After flagellar extraction, the purified flagellin showed an apparent molecular mass of 46 kDa with an isoelectric point of 3.6. Sugar staining, mild periodate oxidation and beta-elimination experiments showed that the flagellin was glycosylated and that the 21E7-B12 epitope was located in the sugar moiety. Amino acid composition showed that the flagellar filament protein contained a high percentage of serine and threonine, while proline was absent. The first 23 residues of the N-terminal were determined and sequence homology with other flagellins was found.

Expression of the Drosophila retrovirus gypsy as ultrastructurally detectable particles in the ovaries of flies carrying a permissive flamenco allele.

The endogenous retrovirus gypsy is controlled by the Drosophila gene flamenco (flam). New insertions of gypsy occur in any individual Drosophila if its mother is homozygous for the flam1 permissive allele and contains functional gypsy proviruses. The ovaries of flam1 females also contain high amounts of gypsy RNAs. Unexpectedly however, gypsy derepression does not occur in the flam1 female germ-line proper but in the somatic follicular epithelium of the ovary. Since extracts from these females are able to efficiently infect the germ-line of a strain devoid of active gypsy proviruses, we assume that a similar kind of germ-line infection, which would occur inside the flam1 females themselves, could be required for gypsy insertions to occur in their progeny. This hypothesis was confirmed by electron microscopy observations showing that non-enveloped intracytoplasmic particles containing gypsy RNAs accumulate in the apical region of the flam1 follicle cells, close to specific membrane domains to which the gypsy envelope proteins are targeted, whereas both are absent in the flam+ controls. Low amounts of similar virus-like particles were also observed in flam1 oocytes, but it is not yet known whether they entered passively or as a result of membrane fusion. This is the first report of the beginning of a retrovirus cycle in invertebrates and these observations should be taken into account when explaining the maternal effect of the flamenco gene on the multiplication of gypsy proviruses.

Biochemical and molecular consequences of massive mitochondrial gene loss in different tissues of a mutant strain of Drosophila subobscura.

In the studied mutant strain of Drosophila subobscura, 78% of the mitochondrial genomes lost >30% of the coding region by deletion. The mutations was genetically stable. Despite this massive loss of mitochondrial genes, the mutant did not seem to be affected. Distribution of the two genome types, cell levels of mitochondrial DNA, steady-state concentrations of the mitochondrial gene transcripts, mitochondrial enzymatic activities, and ATP synthesis capacities were measured in the head, thorax, and abdomen fractions of the mutant strain in comparison with a wild type strain. Results indicate that the deleted genomes are detected in all fractions but to a lesser extent in the male and female abdomen. In all fractions, there is a 50% increase in cellular mitochondrial DNA content. Although there is a decrease in steady-state concentrations of mitochondrial transcripts of genes affected by deletion, this is smaller than expected. The variations in mitochondrial biochemical activities in the different fractions of the wild strain are upheld in the mutant strain. Activity of complex I (involved in mutation) nevertheless shows a decrease in all fractions; activity of complex III (likewise involved) shows little or no change; finally, mitochondrial ATP synthesis capacity is identical to that observed in the wild strain. This latter finding possibly accounts for the lack of phenotype. This mutant is a good model for studying mitochondrial genome alterations and the role of the nuclear genome in these phenomena.

Exportation of mouse vas deferens protein, a protein without a signal peptide, from mouse vas deferens epithelium: a model of apocrine secretion.

Mouse vas deferens protein (MVDP) is a major androgen-dependent protein of deferential fluid. It is specifically expressed in the epithelium of the mouse vas deferens. Its amino acid sequence as deduced from the nucleotidic sequence of its cDNA does not possess a signal sequence characteristic of secretory proteins. In vitro, transcription of MVDP cDNA followed by translation of mRNA in the rabbit reticulocyte system, in the absence or the presence of microsomes, demonstrated that there was no internalization of MVDP into microsomes that could protect it from degradation by proteinase K; this confirmed the absence of signal sequence. Moreover, MVDP has its NH2-terminus blocked. To understand how MVDP can be exported, its ultrastructural distribution and secretion process were analyzed by means of electron microscopy. Immunolocalization of MVDP revealed that it was distributed in the whole cytoplasm; it was never detected in the lumen of endoplasmic reticulum, Golgi apparatus, or vesicles but was abundant in apical protrusions and in the fluid, where it was associated with cellular material undergoing degradation. These data clearly demonstrated that exportation of MVDP into the luminal fluid does not occur in the classical manner for secretory proteins but rather involves an apocrine secretion process.

Seasonal variations in the ultrastructure and production of androgen-dependent proteins in the seminal vesicles of a Saharian rodent (Psammomys obesus).

The effects of seasonal variations and experimental deprivation and substitution of androgen in the seminal vesicles of the Saharian rodent Psammomys obesus were investigated. Cytological studies showed that, during the breeding season, epithelial cells had large amounts of rough endoplasmic reticulum (RER) and substantial apocrine secretion. During the non-breeding season, RER cisternae were no longer expanded and apocrine secretion was rare. Castration during the breeding season was followed by regression of the RER and the disappearance of apocrine secretion. Treating castrated animals and animals in the non-breeding season with testosterone for 15 days caused reactivation. Eight major proteins with molecular weights of 120, 78, 67, 41, 37, 34, 21 and 14.4 kDa were present in homogenates during the breeding season; six (92, 41, 36, 35, 21 and 14.4 kDa) were found in seminal vesicle secretions. During the non-breeding season, the very large amounts of the 21 kDa protein were greatly reduced; conversely the 45 kDa protein increased. Electrophoretic patterns of homogenates from animals castrated during the breeding season showed eight proteins differentially induced (37, 34, 21 and 18 kDa) or repressed (67, 45, 38 and 35 kDa) by testosterone, of which the 21 kDa protein decreased most dramatically after castration. The effects of castration were reversed by the administration of testosterone. During the non-breeding season, the synthesis of the four induced proteins was stimulated by testosterone treatment; conversely that of the 45 kDa protein was suppressed.

Germ-line expression of a functional LINE from Drosophila melanogaster: fine characterization allows for potential investigations of trans-regulators.

The I factor (IF) is a functional non-viral retrotransposon, or LINE, from Drosophila melanogaster. It is mobilized in the germ-line of dysgenic SF females during I-R hybrid dysgenesis. In previous papers (Lachaume et al., Development 115: 729-735, 1992; Lachaume and Pinon, Mol. Gen. Gen. 240: 277-285, 1993) we used a transgenic fusion between the 5' part of the IF and the lacZ gene to characterize IF expression and its regulation. This I-lacZ transgenic fusion expresses beta-galactosidase activity during oogenesis. We established a Drosophila line bearing four transgenic insertions (the 4I-lacZ line) and got new insights about IF expression: (1) I-lacZ expression is proportional to the copy number of transgenes present in the genome, (2) the expression occurs just before or when meiosis begins, (3) this expression seems to be subjected to a variegation effect within the germ-line cells, (4) the transgenic activity is mainly directed toward the decondensed chromatin of nurse cells. The close relationship between I factor expression and oogenesis led us to investigate the role played by genes expressed during oogenesis on I factor expression. We present recent data indicating that mutants which interfere with oogenesis can also affect I factor expression. From this data we propose an original screen using the 41-lacZline to detect identified mutations which also affect I factor expression.

Tissular distribution of heteroplasmy and ultrastructural studies of mitochondria from a Drosophila subobscura mitochondrial deletion mutant.

A mutant strain of Drosophila subobscura possesses two mitochondrial genome types: a minority population (20%) identical to the wild strain mtDNA (15.9 kb), and a largely predominant population (80%) of shorter genomes (10.9 kb), presenting a deletion of more than 30% of its coding region. Study of tissular distribution of heteroplasmy shows it to be identical--about 80%--in the head (nervous tissue) and thorax (muscles). On the other hand, a lower percentage (64%) is observed in the ovaries. The strain is apparently unaffected despite this massive loss of genes, coding for four tRNA and for complex I and III subunits. Contrary to observations of similar situations in man, the mutant strain shows no accumulation or structurally abnormal mitochondria. Furthermore, cytochemical studies fail to detect mitochondria devoid of cytochrome oxidase activity (COX-). Finally, mitoribosome populations are identical in mitochondria from both strains. These results suggest that, in the mutant strain, there are no mitochondria containing deleted genomes only: heteroplasmy would thus be intramitochondrial.

In vitro androgenic induction of a major protein in epithelial cell subcultures from mouse vas deferens.

Pure epithelial cell cultures, obtained from primary culture of vas deferens tissue collected from 20- to 30-day-old mice, were amplified by subculturing the cells over 3T3 feeder layer in a serum-free defined medium. Adhesion and proliferation of epithelial cells did not require androgens, but a minimal concentration of 5.10(-7) M hydrocortisone. In that system, epithelial cells expressed cytokeratin but failed to produce the tissue specific mouse vas deferens protein (MVDP) in response to androgens. Various culture procedures and medium compositions were assayed for induction of MVDP expression. Culture onto microporous membrane inserts, which allow polarization of cells, is absolutely required for androgenic induction of MVDP. Androgen action did not require the presence of hydrocortisone, insulin, triiodothyronine, pituitary extracts, epidermal growth factor and acetylcholine. A minimal supplemented medium was then defined in which the expression of MVDP by epithelial cells in response to androgens was dose dependent. It has also been shown that this response at each concentration of dihydrotestosterone was heterogeneous at individual cell level. Highly reproducible results were obtained from epithelial cell cultures between 8th to 16th passages, showing that subcultured cells have maintained their ability to differentiate and express specialized functions.

A newly described role for protein glycosylation: starved yeast cells absorb their under-glycosylated secreted xylanase faster than the glycosylated enzyme.

The yeast Cryptococcus albidus secretes a highly glycosylated xylanase into the culture medium, when grown in presence of xylan, but addition of tunicamycin to the medium results in the formation of an underglycosylated xylanase. Both types of enzyme preparation were incubated with starved yeast cells. Assimilation of the xylanases by the cells over a period of time was followed by electron microscopy using immunolocalization with anti-xylanase antibodies coupled to gold-labelled protein A. Electron micrographs showed that the glycosylated enzyme mostly remained attached to the cell wall surface, while the underglycosylated enzyme not only surrounded the cell wall but was also present in the hyaloplasm, indicating its assimilation by the cells. These experiments indicate that the carbohydrate moiety of the xylanase protects the enzyme from its assimilation by the cells producing it.

Mitochondrial development during Drosophila oogenesis: distribution, density and in situ RNA hybridizations.

The changes in distribution and density of mitochondria and the level of mitochondrial RNA during Drosophila oogenesis were studied simultaneously in the 3 cell types ie follicle cells, nurse cells and oocyte, making up the egg chamber. Up to stage 6, mitochondrial density (mitochondrial and cellular areas ratio) was elevated and increased similarly in both follicle and nurse cells. Thereafter the mitochondrial density of follicle cells continued to increase and that of the nurse cells declined markedly while the nurse cell mitochondria assembled in dense groups and decreased in size. This can be related to a transfer of nurse cell cytoplasm, including mitochondria, to the oocyte. In the oocyte from stage 4 to stage 7 we observed a significant decrease of the mitochondrial density due to the absence of mitochondrial biogenesis. Then the cytoplasm transfer caused mitochondrial density to increase up to the level found in the nurse cells at the end of oogenesis. The mature oocyte contains enough mitochondria to supply 15,000 somatic cells. Our results strongly suggest that the variations in size, distribution and density of mitochondria relate to the particular energetic requirements of the different cell types during the first half of oogenesis. Later they relate to the developmental requirements of the nurse cells and the oocyte, in particular the storage of mitochondria in the oocyte. The level of mitochondrial RNA was studied through in situ hybridization. Throughout oogenesis the follicle and nurse cell RNA evolved similarly. Up to stage 9, there was no change in RNA densities in these cells, suggesting a correlation with the cell volume and/or the nuclear DNA content. Thereafter the cellular RNA concentration declined rapidly. In the oocyte the RNA concentration evolved differently especially from stage 10 to the end, the RNA density being stabilized. This can be related to the injection of nurse cell mitochondria, followed by their assignment to reserve status. Our results suggest that the mt RNA density is under extramitochondrial control mechanisms.

20-OH-ecdysone regulates 60 C beta tubulin gene expression in Kc cells and during Drosophila development.

Cultured Kc cells of Drosophila melanogaster are sensitive to the insect moulting hormone, 20-hydroxy-ecdysone (20-OH-E). Morphological changes of Kc-treated cells were observed and electron microscopic analysis of pseudopodia shows a large increase in the number of microtubules, all arranged in the same orientation. The 60 C beta tubulin gene which is expressed only in 20-OH-E-treated cells encodes a 2.6-kb mRNA which is essentially cytoplasmic and polyadenylated. The corresponding premessenger is 7 kb in length and is absent in untreated cells. Two peaks of expression of the 60 C beta tubulin gene are observed during Drosophila development: at midembryogenesis (stage 8-13 h) and at the late third instar larvae-early pupae stage. By use of the Ecdysone 1 mutant, 60 C beta tubulin gene expression was demonstrated to be regulated in part by 20-OH-E during Drosophila development. Through these two complementary biological models of study, the mode and role of beta tubulin gene regulation are discussed.

Effect of tunicamycin on xylanase secretion in the yeast Cryptococcus albidus.

The yeast Cryptococcus albidus secretes a glycosylated xylanase (48 kDa) in the culture medium in response to beta-methylxyloside as inducer. Addition of tunicamycin to the medium results in the formation of a modified xylanase (40 kDa) which is depleted in carbohydrate content and whose enzymatic activity is 2.5 times less than that of the glycosylated xylanase. The secretion of xylanase was followed under both conditions by pulse-chase experiments. The half-time of secretion of the glycosylated and nonglycosylated forms was 5 and 2 h, respectively. Cell-associated xylanase activity was not detected when the cells were treated with the antibiotic. The absence of cell wall-associated xylanase, after tunicamycin treatment, was confirmed by immunolocalization with anti-xylanase antibodies at the electron microscopic level. The results suggest that the interactions of carbohydrate moiety within the cell wall retarded the secretion of the enzyme to the medium.

Comparison between two chromogenic substrates for revealing an immunoperoxidase reaction of human metaphase chromosomes.

To study the binding of an antiadenosine serum to human chromosome DNA, two types of chromogenic reagents were compared. The procedure is as follows: lymphocytes with metaphase chromosomes were spread on slides; some slides were irradiated by UV; all slides were then incubated with antiadenosine rabbit serum and then with antirabbit sheep serum labelled with peroxidase; the latter was revealed in the classical manner by 3,3'-diaminobenzidine (DAB), or, alternatively, by p-phenylenediamine plus pyrocatechol (PPD-PC). The present study shows that the results obtained with PPD-PC were equivalent, if not superior, to those obtained with DAB. In the case of weaker reactions, results with PPD-PC were superior. Furthermore, this reagent has the major advantage of being noncarcinogenic.

[Preliminary study of stages of human meiosis in spermatocytes and ovocytes (author's transl)]. / Etude preliminaire de stades de la méiose humaine dans les spermatocytes et les ovocytes.

Human meiotic chromosomes, from spermatocytes and ovocytes, are described after observations of whole mount preparations under E.M. Small testicular and ovarian fragments are put in distillated water, then macerated; the cell suspension is spread on the surface of sheet copper grids covered with formvar plus collodion films. After dehydratation interesting stages are selected under L.M. before observations under E.M. Zygotene and pachytene are the most common stages. During pachytene the chromomeres are well individualized; the synaptonemal complex may be observed; chromatin fibers connect the chromosomes to nuclear pores, interchromosomal fibers joint the bivalents. Zygotene and pachytene bivalents are very similar in the male and the feminine germ cells.

[Karyotypes, supernumerary chromosomes, rudimentary parthenogenesis and polyploidy in 2 species of the genus Phronima (Crustacea, Amphipoda)]. / Caryotypes, chromosomes surnuméraires, parthénogenèse rudimentaire et polyploidie chez deux espèces du genre Phronima (Crustaces, Amphipodes)

The diploid number of Phronima sedentaria and P. atlantica is 30, all the chromosomes are metacentric or submetacentric; the caryotypes of these two species are compared with those of other Amphipods. Some individuals of each species have a supernumerary chromosome. This element, found in both sexes, remains as an univalent at meiosis and goes earlier to one pole. In some cases nonfertilized ova undergo segmentation; this rudimentary parthenogenesis stops quickly and is partly related to polyploidization.