Correction of mouse ornithine transcarbamylase deficiency by gene transfer into the germ line

The sparse fur with abnormal skin and hair (Spf-ash) mouse is a model for the human X-linked hereditary disorder, ornithine transcarbamylase (OTC) deficiency. In Spf-ash mice, both OTC mRNA and enzyme activity are 5% of control values resulting in hyperammonemia, pronounced orotic aciduria and an abnormal phenotype characterized by growth retardation and sparse fur. Using microinjection, we introduced a construction containing rat OTC cDNA linked to the SV40 early promoter into fertilized eggs of Spf-ash mice. The expression of the transgene resulted in the development of a transgenic mouse whose phenotype and orotic acid excretion are fully normalized. Thus, the possibility of correcting hereditary enzymatic defect by gene transfer of heterologous cDNA coding for the normal enzyme has been demonstrated.     

Site-specific integration of plasmid pSAM2 in Streptomyces lividans and S. ambofaciens

Summary Streptomyces ambofaciens strain ATCC23877 contains the 11.1 kb plasmid pSAM2 stably integrated into its chromosome. This plasmidic sequence is able to loop out and to be transferred at high frequency to S. lividans where it is found simultaneously as both free and integrated plasmid. When a UV derivative of strain ATCC23877 (strain ATCC15154) is used, the resident copy of pSAM2 can be transferred to S. lividans, but only the integrated form is found in this strain. In both cases, the integration occurs at a unique chromosomal region through the same plasmidic integration site as that in strain ATCC23877. The resident copy of strain ATCC15154 can also be transferred at low frequency to S. ambofaciens DSM40697 (devoid of any pSAM2 sequence). In this case, as several copies of pSAM2 are integrated, the integration pattern is complicated. Integration of a complete pSAM2 sequence in this strain occurs in a region that hybridizes with the integration zones of S. lividans and of S. ambofaciens strain ATCC23877. Comparison of the cloned integration zone of S. lividans before and after the integration event showed that the restriction pattern of the resident pSAM2 in strain ATCC15154 is similar to that of the free form of pSAM2 found naturally in another UV derivative of strain ATCC23877 (strain JI3212).     

Attempts to transform Zea mays via pollen grains

Summary An attempt was made to transfer two sorts of DNA into maize via pollen grains. Controls for both pollen quality and DNA behaviour during the transformation experiments were included. When genomic DNA was used, no transformants were observed among the 1805 seeds screened. With plasmid DNA (harbouring the gene expressing kanamycin resistance in plant cells), three plants with kanamycin resistance were observed among the 1723 seeds screened, although no molecular evidence of transformation was obtained. Experiments indicated that under the conditions used, DNA was being degraded by both pollen and stigma nucleases. Consequently, we attempted to determine protocols which would inhibit these nuclease activities in order to preserve DNA integrity during transformation experiments, thus allowing fertilization. We found that a classic germination medium supplemented by 300 or 600 mM KNO₃, or 20% PEG₁₅₅₀ satisfied all these conditions.Abbreviations BK Brewbaker and Kwack medium - BKS15 Brewbaker and Kwack medium containing 15% (w/v) sucrose - EDTA ethyldiaminetetraacetic acid - FCR fluorochromatic reaction - FP fertilization percentage - PEG polyethylene-glycol     

Dissection of the bifunctional ARGRII protein involved in the regulation of arginine anabolic and catabolic pathways.

ARGRII is a regulatory protein which regulates the arginine anabolic and catabolic pathways in combination with ARGRI and ARGRIII. We have investigated, by deletion analysis and fusion to LexA protein, the different domains of ARGRII protein. In contrast to other yeast regulatory proteins, 92% of ARGRII is necessary for its anabolic repression function and 80% is necessary for its catabolic activator function. We can define three domains in this protein: a putative DNA-binding domain containing a zinc finger motif, a region more involved in the repression activity located around the RNase-like sequence, and a large activation domain.     

Selectivity of juxtaposition between cup-shaped lactotrophs and gonadotrophs from rat anterior pituitary in culture

Summary Semi-thin sections of three-dimensional reaggregates from adult female rat pituitary, cultured in serum-free defined medium, were stained for prolactin, gonadotropin, thyrotropin, growth hormone and S-100, using the double immunolabelling technique. The frequency of juxtaposition between lactotrophs and gonadotrophs was enumerated and compared with the expected frequency at random distribution of polygonal cell profiles in a hexagonal configuration. The proportions of lactotrophs and gonadotrophs in the aggregate sections were determined using stereometrical analysis. The observed frequency of juxtaposition did not differ significantly from the expected frequency. Hence, no reason was found to assume a selective adhesion between lactotrophs and gonadotrophs in adult female rat pituitary reaggregates. A constant proportion of lactotrophs was found to meet the criteria of a cup-shaped morphology, and 70%±9% (mean ±S.D.) of these so-called cupshaped lactotrophs were found to be juxtaposed at their concave side to gonadotrophs. Administration of 0.01 nM 17-oestradiol to the culture medium resulted in a significant reduction of the proportion of cup-shaped lactotrophs but did not affect the selectivity of juxtaposition to gonadotrophs. The selectivity of juxtaposition between cup-shaped lactotrophs and gonadotrophs may be the morphological correlate of the functional relationship between these cells, which are known to be involved in an intra-pituitary paracrine communication system.     

Cell types of the pars distalis of the hedgehog (Erinaceus europaeus L.) adenohypophysis: cytological, immunocytochemical and ultrastructural studies. 4. Thyrotropic cells

Staining and histochemical methods, immunofluorescence and electron microscopy were used to individualize the thyrotropic cells in the adenohypophysis of the non-hibernating hedgehog. One cell type was differentiated; its characteristics at the light and electron-microscopic levels were presented. Immunofluorescence has confirmed the functional significance of this cell type and the validity of the denomination of 'thyrotropic cells'.     

Occurrence of a microcanalicular system within the ossicles and dermal tissue of Asterias rubens and Marthasterias glacialis (Echinodermata: Asteroidea)

Summary A microcanalicular network is demonstrated within the ossicle stroma and the dermal tissue of two asteroid species. Microcanaliculi are presumed to be mesodermal structures. They consist of convoluted tubular ducts lined by epithelial cells associated with scattered basiepithelial nervous processes. Such a microcanalicular system has not been reported previously from any echinoderm species. Its discovery in asteroids entails some conceptual changes, especially considering the physiology of the body wall.Research assistants of the National Fund for Scientific Research (NFSR, Belgium)