Dominique Hue

Cloning and expression of cDNA encoding ovine trophoblastin: its identity with a class-II alpha interferon

The cDNAs encoding ovine trophoblastin (oTP) were isolated from an ovine embryo cDNA lambda gt 11 library by screening with a synthetic 29-mer oligodeoxynucleotide corresponding to amino acid (aa) residues 34 to 43 of oTP. The cDNA contained an open reading frame of 595 bp and the deduced amino acid sequence indicates a protein precursor of 195 aa. Nucleotide and amino acid sequence comparisons establish that oTP shares extensive homology with alpha-interferon (IFN-alpha) but is more closely related to the IFN-alpha sII subfamily. When the oTP cDNA was cloned into an eukaryotic expression vector and transfected in monkey COS cells, a high level of antiviral activity was detected. RNA blot analyses of total RNA reveal that the oTP-coding gene is expressed during a relatively short period (eleven to 21 days). The abundant expression of oTP mRNA corresponds closely to the time at which the embryo acts to extend luteal lifespan. RNAs homologous to oTP were also detected in goat and cow embryos at equivalent periods of their development, but not in the pig.

Amino terminal sequence of the precursor of ovine β-lactoglobulin

Abstract Ovine mammary gland mRNAs were translated in a wheat-germ cell-free system in the presence of radioactive amino acids. Automated Edman degradation performed on α-lactalbumin isolated by immunoprecipitation from the mixture of radiolabelled lactoproteins showed the occurrence of an hydrophobic 19 residues long amino terminal extension. The pre-protein represents the primary translation product since the amino terminal methionyl residue was found to be donated by initiator Met-tRNA i Met . Comparison of the signals of ovine α-lactalbumin and hens egg white lysozyme, two homologous proteins which are thought to be derived from a common ancestor, suggests that the signal region has evolved at least as rapidly as the remaining part of the polypeptide chain.

Expression of a biologically active ovine trophoblastic interferon using a baculovirus expression system.

Ovine trophoblast protein (oTP) an embryonic interferon, which plays a key role in maternal recognition of pregnancy, has been expressed in insect cells using a baculovirus expression system. A cDNA coding for oTP was inserted downstream of the strong polyhedrin promoter. Cells infected with recombinant virus produced biologically active oTP and greater than 90% was secreted into the culture medium during infection. High amount of antiviral activity were produced (up to 5 x 10(5) IU per ml of culture medium). Recombinant oTP (roTP) was purified by immunoaffinity chromatography and found to be identical to authentic oTP with respect to molecular mass and N-terminal amino acid sequence.

ENZYMATIC PROCESSING OF PRECURSORS OF OVINE LACTOPROTEINS BY MAMMARY MICROSOMAL MEMBRANES AND A DEOXYCHOLATE-SOLUBLE EXTRACT FROM ROUGH MICROSOMES

The signal hypothesis first proposed in [l] and further refined [2,3] has provided an attractive model to explain adequately the mechanism involved in selecting specific mRNAs for translation on bound polyribosomes at the early stage of protein secretion. Analyses of miscellaneous secretory proteins synthesized in various cell-free systems have shown that the polypeptide chains thus manufactured contain hydrophobic amino terminal extensions containing up to 30 amino acid residues [4,5]. This hydrophobic segment, called the ‘signal’, interacts with the endoplasmic reticulum (ER) membranes, thus providing the topological conditions for the vectorial discharge of the nascent polypeptide chain into the ER lumen. Subsequently the ‘signal’ is selectively removed through proteolytic cleavage, which does occur before completion of the chains, since the processing of preproteins can only be achieved when microsomal membranes are already present in the cell-free translation system [2,3,6-l 11. In order to investigate the mechanism of protein secretion in the lactating mammary gland, the primary structures of the 6 major lactoproteins (a,r-, crs2-, /3and K-caseins, @-lactoglobulin and a-lactalbumin) synthesized in a cell free system were analysed. The in vitro translation products were found to contain amino terminal extensions of 15 amino acid residues for the first three caseins and of 21, 18 and 19 for the three latter proteins, respectively [5,12,13].

Amino terminal sequence of porcine pre-β-lactoglobulin. Comparison with its ovine counterpart

Abstract Porcine mammary gland mRNAs were translated in a wheat germ cell-free system in the presence of radioactive amino acids. Automated Edman degradation performed on β-lactoglobulin isolated by immunoprecipitation from the mixture of radiolabeled lactoproteins showed the occurrence of a hydrophobic amino terminal extension made up of 18 amino acid residues. Comparison of the amino terminal sequences of porcine and ovine pre-β-lactoglobulins revealed a high degree of homology in the signal peptide region. This suggests that the efficient transfer of that protein across the endoplasmic reticulum membrane requires the structural integrity of the transient amino terminal extension.

Spermatogenèsein vitro: une nouvelle voie de recherche

RésuméNous avons mis au point au laboratoire deux systèmes de coculture permettant à certaines étapes de la différenciation de cellules germinales mâles mâles de rat et de souris de s’effectuer in vitro. Le premier système est une coculture de spermatocytes pachytènes de rats adultes et de cellules de Sertoli provenant d’animaux de 20 jours. Dans le second système de petits fragments de tubes séminifères sont ensemencés. De cette façon les jonctions intercellulaires qui sont présentsin vivo sont mieux maintenuesin vitro; de plus chaque type de cellule germinale présent dans les fragments de tubules au moment de l’ensemencement peut être étudié. Nos résultats ont montré que les deux divisions méiotiques peuvent se dérouler au cours d’une période de 2 à 4 semaines de culture. Ces résultats devraient avoir des applications dans différents domaines: recherche fondamentale, toxicologie, études cliniques et biotechnologies. Cependant, quelques améliorations et vérifications sont encore nécessaires pour établir la faisabilité et l’innocuité de ces procédures.AbstractStudies in our laboratory have aimed to settle two culture systems allowing some steps of rodent spermatogenic cell differentiation to occurin vitro. The first system is a coculture of pachytene spermatocytes from adult rats together with Sertoli cells from 20-day-old animals. In the second system, small pieces of spermatogenic tubules are seeded. In that way the cell-cell junctions which are presentin vivo are better maintainedin vitro; in addition every type of germ cell present in the tubule segments at the time of seeding can be studied. Our results have shown that the two meiotic divisions or even most of the meiotic process can occur over a 2 to 4 week-culture period. These results should have applications in basic research, toxicology, clinical studies and biotechnology. However, for these latter, some improvements and verifications are still needed in order to assess the feasibility and the innocuity of these procedures.