Cystic fibrosis typing with DNA probes and screening for ΔF508 deletion in families from Southern France

Summary A sample of 235 individuals from 49 French cystic fibrosis (CF) families with at least one living affected child was typed with probes for restriction fragment length polymorphisms (RFLPs) known to be linked to the CF gene, and was screened for the ΔF508 mutation. Using a combination of six probes, 44 out of the 49 families were sufficiently informative to enable prenatal diagnosis or carrier determination. As in many other populations, linkage disequilibrium was found between the CF locus and the haplotype B (XV2c: allele 1; KM19: allele 2), which accounts for about 78% of CF chromosomes in our families. The ΔF508 deletion was present in 64.3% of CF chromosomes.     

Somatic hybrid plants produced by electrofusion between dihaploid potatoes: BF15 (H1), Aminca (H6) and Cardinal (H3)

In order to regenerate somatic hybrids, mesophyll protoplasts from a dihaploid potato, BF15 (H1), were electrofused with those from two other dihaploid clones, Aminca (H6) and Cardinal (H3). Determination of the ploidy level by flow cytometry showed that 10% of plants regenerated from the fusion experiment with BF15 + Aminca were diploids, 14% triploids, 63% tetraploids and very few were mixoploids or had a higher ploidy level. Using morphological markers and vigour in plant growth, we were able to recover a total of 24 somatic hybrid plants, respectively 20 and 4 hybrids (accounting for 12% and 13% of regenerants) from the fusions BF15 + Aminca and BF15 + Cardinal. Most of the somatic hybrids were at the expected tetraploid level (2n=4x=48). The hybrid nature was confirmed by examining isoenzyme patterns for malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICD).     

Targeting and fusion in vesicular transport

Vesicular transport of proteins and lipids between distinct subcellular compartments is directly responsible for generating and maintaining the structure of the organelles of the secretory and endocytic pathways in eukaryotic cells. Rapid advances in a variety of experimental systems have resulted in the identification of molecules involved in late steps of the transport process. This article presents a general paradigm for vesicular fusion and reviews the available experimental evidence.     

Optimization of growth conditions for the production of proteolytically-sensitive proteins in the periplasmic space of Escherichia coli

Summary The expression of many secreted recombinant proteins in Gram-negative bacteria is limited by degradation in the periplasmic space. We have previously shown that the production of protein A--lactamase, a secreted fusion protein highly sensitive to proteolysis in Escherichia coli, can be increased in mutant strains deficient in up to three cell-envelope-associated proteolytic activities. In this work we investigated the effect of fermentation conditions on suppressing any residual proteolytic activity in various protease-deficient strains. Optimal production of the fusion protein was observed in cells grown under mildly acidic conditions (5.5pH6.0) and at low temperatures. These conditios were shown to specifically decrease the rate of proteolysis. In addition, a further increase in production was observed in cultures supplemented with 0.5 to 0.75 mM zinc chloride. This may relate to the inhibition of a cell envelope protease by Zn²⁺ ions. Offsprint requests to: G. Georgiou     

Comparison of the amino acid sequences of human protamines HP2 and HP3 and of intermediate basic nuclear proteins HPS1 and HPS2. Structural evidence that HPS1 and HPS2 are pro-protamines

Two intermediate nuclear basic proteins HPS1 and HPS2 were isolated from human sperm. They were characterized by their electrophoretic mobility in acid-urea gels, their amino acid composition, and their peptide maps after digestion by endoproteinase Lys-C and by endoproteinase Glu-C. Their amino-terminal amino acid sequences have also been determined. The structural data thus obtained suggest that HPS1 and HPS2 are precursors of human protamines HP2 and HP3.     

Mitogenic effect of PMA + IL 2 on subpopulations of corticoresistant thymocytes

The proliferative response of subpopulations of corticoresistant thymocytes (CRT) to phorbol-12-myristate-13-acetate (PMA) + interleukin 2 (IL 2) was investigated. Thymocyte subpopulations were selected by the indirect "panning" technique, and their purity was checked by cytofluorometry. Microcultures were set up with an optimal concentration of PMA, EL4 supernatant, or pure IL 2 obtained by recombinant DNA technology (r-IL 2) in the presence or in the absence of accessory splenic adherent cells (SAC). Under these conditions, only the Lyt-2+ CRT proliferated, and this response was IL 2-dose-dependent and was increased by accessory cells. When the calcium ionophore A23187 was added to the cultures, the proliferation of L3T4+ CRT was greatly increased. These results were confirmed by cultures at limiting dilution of positively selected Lyt-2+ and L3T4+ subpopulations of CRT at optimal concentrations of PMA, r-IL 2, A23187, and accessory cells. These results are consistent with the idea that two signals are necessary to activate L3T4+ CRT, whereas only IL 2 is necessary for PMA-induced proliferation of Lyt-2+ CRT. Finally, unlike the case of lectin-induced proliferation of Lyt-2+ and L3T4+ CRT, the presence of accessory cells or cell-cell contact is important for optimal response to PMA + IL 2.     

Oligonucleotides covalently linked to intercalating agents. Influence of positively charged substituents on binding to complementary sequences

A pentamethylene chain was used to covalently link the 3'-phosphate of oligothymidylates to the 9-amino group of an acridine derivative. Positively charged substituents were further attached to the 3'-phosphate group to form 3'-phosphotriesters. These molecules form specific complexes with poly(rA) which involve the formation of a number of A X T base pairs equal to that of thymines in the oligonucleotide. Absorption changes induced in the acridine absorption bands are similar to those expected upon intercalation of the acridine dye between A X T base pairs. The acridine covalently linked to the 3'-phosphate strongly stabilizes the complexes formed with poly(rA) as compared with the corresponding unsubstituted oligodeoxynucleotide. The presence of a positively charged substituent on the 3'-phosphate together with the acridine dye further enhances the interaction. The effect of salt concentration on complex stability depends on the number of negatively charged phosphate groups of the oligodeoxynucleotide and on the nature of the substituents borne by the 3'-phosphate group. When the oligothymidylate is substituted by an acridine dye, the stability of the poly(rA) complexes increases when salt concentration increases. If an additional positively charged substituent is present on the 3'-phosphate group, stability decreases when salt concentration increases for the shortest oligonucleotide (trimer) and increases with longer oligonucleotides. Thermodynamic parameters have been calculated from the concentration dependence of melting temperatures.     

The isolation and genetic analysis of aCaenorhabditis elegants translocation (szT1) strain bearing an X-chromosome balancer

A single X-chromosome balancer-bearingCelegans ♂+, as a founder of a strain (AF1), was isolated directly from Fl progeny of irradiated+dpy-8unc-3/lon-2++ hermaphrodites on the basis of the absence of recombinant F2 categories. The balancer chromosome (Bal-X-1) suppresses recombination over a two-thirds section of the X chromosome (between genesdpy-8 andlet-2) and is associated with a reciprocal translocation between linkage groups (LG) X and I. Animals homozygous for the translocation (szT1(X:1)) are nonviable. Hermaphrodites heterozygous for the translocation segregate male selfprogeny at a frequency of 0.08-0.12.Bal-X-l carries the marker mutationlon-2(e678) and can be detected cytologically. This balancer chromosome proved useful for rnaimaininga number of X-linked lethal mutations and deficiencies inC. elegans.