The HLA-D system: At least two loci and four distinct phenotypic traits per haplotype. Introduction to component typing in families and population by primed lymphocyte typing

Using a number of intrafamilial PLTs raised against identical HLA haplotypes it has been possible to construct a model in an informative family defining the HLA-D region as a genetic system. This system consists of at least two regions separated by a recombination between HLA-D and GLO. In relation to the site of recombination, a minimum of one centromeric and three telomeric components can be identified per haplotype.—Fourteen PLTs raised and defined within the family were subsequently tested in a Caucasian population (n=84) and in 13 unrelated, complete families.—It is concluded that the hypothetical model proposed for the HLA-D region as a genetic system of linked loci, coding at the cell surface for associated but distinct components (at least four per haplotype), allows for typing of the components of the HLA-D system of any given haplotype. Serological typing of HLA-D components should, in the near future, provide a more convenient way of establishing component phenotypes than the present use of primed lymphocyte typing reagents. Among the components isolated, some have a high association with the classic alleles defined either by homozygous typing cells or DR serology. Others form the basis of cross-reactivity but their presence does not interfere with standard typing. Others, however, seem by their mere presence to be responsible for false assignments.—The concept of HLA-D as a genetic system clarifies many of the inconsistencies observed with a one-locus system.Research scientists from INSERM.Research Fellow from the Danish Medical Research Council.Central Blood Bank — Marseille     

Localisation régionale des gènes humains IDHS,MDHS, PGK, α-GAL,G6PD par l'hybridation cellulaire interspécifique

Summary 22 independent man-hamster (HGPRT^–) hybrids using male human cells with balanced reciprocal translocation t(X;2)(p22;q32) were analysed for human genes localized on chromosome 2 (IDH_S, MDH_S), on chromosome X (PGK, GAL, G6PD) and for the different chromosomes in relation with the balanced reciprocal translocation (chr.2, chr.2q^–, chr.Xp⁺).The following results were obtained:The chromosomes 2 and 2q^– are absent in the 22 hybrids.In 9 hybrids, the absence of MDH_S in spite of the presence of the chromosome Xp⁺ indicates that the gene for MDH_S is not localized on this chromosome (or that the gene for MDH_S is not on the segment 2q32-2qter translocated on X).In 14 hybrids, the three markers of X (PGK, GAL, G6PD) and IDH_S are expressed in the presence of the chromosome Xp⁺. This result indicates that the genes for these markers are on Xp⁺ or that the genes PGK, GAL, G6PD are on X without the Xp22-Xter segment, translocated on the chr.2, and that the gene for IDH_S is on the 2q32-2qter segment translocated on X.In 8 hybrids, in the absence of the intact chromosome Xp⁺, the higher percentage of the presence of G6PD (7 hybrids) and the lower percentage of the presence of IDH_S (3 hybrids) are explained by the fact that these hybrids selected in HAT medium had to retain a segment of Xp⁺ bearing the human gene HGPRT. G6PD appeared very close to HGPRT and IDH_S very distant from HGPRT.The study of the different correlations between the presence and the absence of these four markers on Xp⁺ in the different hybrids indicates the following order on the chromosome Xp⁺ from p to q: IDH_s — PGK — GAL — G6PD.

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Groupe INSERM: Directeur J. Frézal

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Groupe CNRS, ER, 149: Directeur J. de Grouchy     

Cellulose acetate electrophoresis of protein kinases: Detection of the active forms using various substrates

Electrophoresis on cellulose acetate strips was used to analyze protein kinases from normal rat liver. In addition to already well-characterized cAMP-dependent protein kinases type I and II and cAMP-independent casein kinases I and II, this method enabled the detection of several supplementary bands corresponding to kinases which were investigated according to their substrate specificity, activation by cAMP, and inhibition by the specific inhibitor of the catalytic subunit of cAMP-dependent protein kinases or by heparin. Using this rapid, sensitive, and resolutive electrophoretic method, different isozyme patterns could be obtained starting from minute amounts of different types of biological material.     

C32 human melanoma cell endogenous lectins: characterization and implication in vesicle-mediated melanin transfer to keratinocytes.

To optimize skin pigmentation in order to help body prevention against UV radiation, the mechanism of melanin pigment transfer from melanocytes to keratinocytes must be elucidated. Melanin transfer to keratinocytes requires specific recognition between keratinocytes and melanocytes or melanosomes. Cell surface sugar-specific receptor (membrane lectin) expression was studied in human C32 melanoma cells, an amelanotic melanoma, by flow cytometry analysis of neoglycoprotein binding as an approach to the molecular specificity. Sugar receptors on melanocytes are mainly specific for alpha-L-fucose. Their expression is enhanced upon treatment by the diacylglycerol analogue 1-oleoyl-2-acetylglycerol, which can induce melanin synthesis in amelanotic human melanoma cells in a dose-dependent manner. Flow cytometry analyses showed a small-sized population of vesicles distinguishable from large cells by their fluorescence properties upon neoglycoprotein binding. Sorting indicated that the small-sized subpopulation is composed of vesicles produced by melanocytic cells. Upon vesicle formation, a selective concentration of sugar receptors specific for 6-phospho-beta-D-galactosides appears in the resulting melanocytic vesicles. Vesicles are recognized and taken up by cultured keratinocytes and a partial inhibitory effect was obtained upon cell incubation in the presence of neoglycoproteins, indicating a possible participation of sugar receptors in this recognition. The validity for such a model to help in understanding the natural melanin transfer by melanosomes is confirmed by electron microscopy, which demonstrates the presence of melanin inside keratinocytic cells upon incubation with melanocytic vesicles.     

The activation process of Arabidopsis thaliana A1 gene encoding the translation elongation factor EF-1α is conserved among angiosperms

In Arabidopsis thaliana, the activation process of the A1 EF-1 gene depends on several elements. Using the GUS reporter gene, transient expression experiments have shown that mutations of upstream cis-acting elements of the A1 promoter, or the deletion of an intron located within the 5 non-coding region, similarly affect expression in dicot or monocot protoplasts. The results reported here strongly suggest that this 5 intron is properly spliced in Zea mays. We show that two trans-acting factors, specifically interacting with an upstream activating sequence (the TEF 1 box), are present in nuclear extracts prepared from A. thaliana, Brassica rapa, Nicotiana tabacum and Z. mays. In addition, a DNA sequence homologous to the TEF 1 box, found at approximately the same location within a Lycopersicon esculentum EF-1 promoter, interacts with the same trans-acting factors. Homologies found between the A. thaliana and L. esculentum TEF 1 box sequences have allowed us to define mutations of this upstream element which affect the interaction with the corresponding trans-acting factors. These results support the notion that the activation processes of A. thaliana EF-1 genes have been conserved among angiosperms and provide interesting data on the functional structure of the TEF 1 box.     

Effect of imazaquin on absorption,translocation, and pattern of distribution of chlormequat chloride in winter wheat

The role of imazaquin in the absorption, translocation, and distribution of chlormequat chloride in CYCOCEL^* CL has been studied in winter wheat. Three treatments were applied to the 5th leaf of the main stem at growth stage 5 (Feekes Large scale): (1)¹⁴C-chlormequat chloride, (2) CYCOCEL^* CL containing¹⁴C-chlormequat chloride, and (3) CYCOCEL^* CL containing¹⁴C-imazaquin. Tracing of the radioactivity was followed in the treated leaf, main stem, tillers, and roots. Results showed that more than 85% of the radioactivity absorbed remained in the treated leaf. Ten days after the application of chlormequat chloride alone, 94.4% of the¹⁴C-chlormequat was found in the treated leaf, 2.9% in the main stem, 1.2% in the tillers, and 1.4% in the root system versus 88.2, 8.2, 2.1, and 1.4%, respectively, for the chlormequat chloride plus imazaquin treatment. It was concluded that imazaquin increases the mobility and the pattern of distribution of chlormequat chloride in the plant.     

Reverse-phase high-pressure liquid chromatography of uridine diphosphate N-acetylmuramyl peptide precursors of bacterial cell wall peptidoglycan

A series of seven different UDP-N-acetylmuramyl peptide precursors of bacterial cell wall peptidoglycan was examined by reverse-phase high-pressure liquid chromatography. Mixtures of these compounds were successfully and rapidly analyzed by using a Waters μBondapak C₁₈ column as a stationary phase and isocratic elutions with 0.05 m ammonium phosphate or formate buffers of appopriate pH. Furthermore, their accurate quantitation could also be readily achieved by reverse-phase high-pressure liquid chromatography. All these techniques should be extremely useful for the purification of these compounds and for a wide range of biochemical studies concerning the cytoplasmic steps of the biosynthesis of peptidoglycan.     

Ans fluorescence and electrokinetic potential of activated lymphocytes by con A or LPS in vivo

The $\text{in vivo}$ effect of Concanavalin A (Con A), or bacterial lipopolysaccharide (LPS) on mouse spleen cell populations was investigated. The membrane fluorescence changes of activated splenic lymphocytes were studied during two weeks after the injection of polyclonal immunogens. Experiments were performed with the hydrophobic fluorescent probe: 1-anilino-8-naphthalene sulphonate (ANS). The kinetic studies further indicated that the course of fluorescence changes may considerably vary depending on both immunogens. These fluorescence intensity changes would be in direct relation to the electrokinetic surface potential changes of activated lymphocytes, as assessed by the electrophoretic mobility analysis. By comparison with the inverse relationship observed in our previous study, it would be concluded that the relation (direct or inverse) between ANS fluorescence and electrokinetic potential depends on the net electrical charge of the antigens used.