Quantitative estimation of chimerism in mice using microsatellite markers

An embryonic stem cell line was established from SV129 mouse blastocysts and used to generate chimeric mice by injection into OF1 blastocysts; 18 out of the 30 resulting offspring appeared chimeric as judged from their coat color patterns, and 3 of the 13 males proved to be germ-line chimeras as they transmitted the SV129 agouti phenotype to all or part of their offspring. The degree of chimerism of these males was evaluated for different tissues using polymorphic microsatellite markers amplified by the polymerase chain reaction. It was shown that these new markers can be effectively used to quantitatively estimate levels of chimerism. The CKMM (creatine kinase, muscle) microsatellite system was used to distinguish the SV129 from the OF1 genotype. In all performed tests, the correlation between DNA ratio and signal ratio, expressed as a base 10 logarithm, was shown to exceed or equal 0.98 for known DNA ratios (SV129/OF1) ranging from 1/99 to 99/1. Linear calibration methods were used to predict the % SV129 DNA of a test sample based on the obtained signal ratio. The accuracy of the prediction was evaluated by performing repeated measurements. Differences among three repeated estimates ranged from 2 to 17% for a given sample. Microsatellite systems should be very useful to monitor chimerism involving strains that can not be discerned with coat color or biochemical markers. This will be particularly important when ES methodology becomes available in species other than mice. © 1993 Wiley-Liss, Inc.     

Two genes present on a transposon-like structure in Lactococcus lactis are involved in a Clp-family proteolytic activity

The lactose-protease plasmid pUCL22 of Lactococcus lactis subsp. lactis strain CNRZ270 contained two inverted copies of IS 1076 flanking a region of 3.7 kb. This internal region was sequenced and found to contain two large open reading frames, ORF1 and ORFP in opposite orientations. ORF1 consists of 2289 bp; the deduced 763-amino-acid sequence is similar to the ATPases of the ClpA family. It contains two well-conserved consensus ATP-binding sites. It was named ClpL. ORFP consists of 930 bp encoding a protein of 310 amino acids. No similarity with any known protein was found in GenBank data for ORFP. Increased ATP-dependent proteolytic activity was detected in extracts from Escherichia coli cells expressing the clpL and ORFP genes.     

Abortion of infection during the Rhizobium meliloti—alfalfa symbiotic interaction is accompanied by a hypersensitive reaction

Nodulation, the organogenetic process resulting from the symbiotic interaction between Rhizobium and legumes, is under the feedback control of the plant. However, the autoregulatory mechanisms controlling root nodule formation are poorly understood. In this paper it is shown that alfalfa can react to infection by its symbiont Rhizobium meliloti by eliciting a defence mechanism similar to the hypersensitive reaction (HR) observed in incompatible plant-pathogen interactions. After the first nodule primordia have been induced, an increasing proportion of infection threads abort in a single or a few root cortical cells in which both symbionts simultaneously undergo necrosis. Autofluorescent, cytochemical and immunolocalization assays revealed that phenolic compounds and proteins associated with defence mechanisms in plants have accumulated in the necrotic cells. These results lead to the proposition that the elicitation of a HR is part of the mechanism by which the plant controls infection and, therefore, regulates nodulation.     

Physical mapping of inhibin β-A in domestic cattle

The gene for the -A subunit of inhibin (INHBA) was assigned to bovine syntenic group U13 by bovine x rodent hybrid somatic cells and the polymerase chain reaction (PCR). A 482-bp PCR fragment was used to clone a 37-kb cosmid. This cosmid was assigned to bovine Chromosome (Chr) 4 (BTA 4) by fluorescence in situ hybridization (FISH). This is the first assignment of a U13 marker to a bovine chromosome. A restriction fragment length polymorphism (RFLP) was detected with PstI within the INHBA cosmid.     

Molecular analysis of the Lactococcus lactis subspecies lactis CNRZ270 bidirectional theta replicating lactose plasmid pUCL22

pUCL22 is the lactose protease plasmid of Lactococcus lactis ssp. lactis CNRZ270. The nucleotide sequence of its replication region Rep22 contains a non-transcribed region, the replication origin, followed by a gene encoding a putative 388-amino-acid protein named Rep22A. The promoter regions of the rep22A and pC194 cat genes share strong similarities and the pUCL22 replicon exerted trans or cis negative control on the pC194 cat gene expression in L. lactis. We suggest that Rep22A binds to its own promoter as well as to the pC194 cat promoter and thus is autoregulated. We show that pUCL22 replicates mainly by a bidirectional theta mechanism in L. lactis, and is representative of a widely distributed replicon family, members of which could be co-resident. We propose that compatibility between these closely related replicons results from minor replication protein modifications coupled with base changes in their respective binding sites, supporting the co-existence of numerous related replicons in lactococcal strains.     

Bioguided Fractionation and Isolation of an Antiplasmodial Saponin from the Roots of Nauclea xanthoxylon (A.Chev.) Aubrév. (Rubiaceae)

The root extract of Nauclea xanthoxylon (A.Chev.) Aubrév. displayed significant 50 % inhibition concentration (IC₅₀s) of 0.57 and 1.26 μg/mL against chloroquine resistant and sensitive Plasmodium falciparum (Pf) Dd2 and 3D7 strains, respectively. Bio-guided fractionation led to an ethyl acetate fraction with IC₅₀s of 2.68 and 1.85 μg/mL and subsequently, to the new quinovic acid saponin named xanthoxyloside ( 1 ) with IC₅₀s of 0.33 and 1.30 μM, respectively against the tested strains. Further compounds obtained from ethyl acetate and hexane fractions were the known clethric acid ( 2 ), ursolic acid ( 3 ), quafrinoic acid ( 4 ), quinovic acid ( 5 ), quinovic acid 3-O-β-D-fucopyranoside ( 6 ), oleanolic acid ( 7 ), oleanolic acid 3-acetate ( 8 ), friedelin ( 9 ), β-sitosterol ( 10a ), stigmasterol ( 10b ) and stigmasterol 3-O-β-D-glucopyranoside ( 11 ). Their structures were characterised with the aid of comprehensive spectroscopic methods (1 and 2D NMR, Mass). Bio-assays were performed using nucleic acid gel stain (SYBR green I)-based fluorescence assay with chloroquine as reference. Extracts and compounds exhibited good selectivity indices (SIs) of >10. Significant antiplasmodial activities measured for the crude extract, the ethyl acetate fraction and xanthoxyloside ( 1 ) from that fraction can justify the use of the root of N. xanthoxylon in ethnomedicine to treat malaria.     

The native F0F1-inhibitor protein complex from beef heart mitochondria and its reconstitution in liposomes

A functional F₀F₁ ATP synthase that contains the endogenous inhibitor protein (F₀F₁I) was isolated by the use of two combined techniques [Adolfsen, R., McClung, J.A., and Moudrianakis, E. N. (1975).Biochemistry 14, 1727–1735; Dreyfus, G., Celis, H., and Ramirez, J. (1984).Anal. Biochem. 142, 215–220]. The preparation is composed of 18 subunits as judged by SDS-PAGE. A steady-state kinetic analysis of the latent ATP synthase complex at various concentrations of ATP showed aV _max of 1.28mol min^–1 mg^–1, whereas theV _max of the complex without the inhibitor was 8.3mol min^–1 mg^–1. In contrast, theK _m for Mg-ATP of F₀F₁ I was 148M, comparable to theK _m value of 142M of the F₀F₁ complex devoid of IF₁. The hydrolytic activity of the F₀F₁I increased severalfold by incubation at 60C at pH 6.8, reaching a maximal ATPase activity of 9.5mol min^–1 mg^–1; at pH 9.0 a rapid increase in the specific activity of hydrolysis was followed by a sharp drop in activity. The latent ATP synthase was reconstituted into liposomes by means of a column filtration method. The proteoliposomes showed ATP-Pi exchange activity which responded to phosphate concentration and was sensitive to energy transfer inhibitors like oligomycin and the uncouplerp-trifluoromethoxyphenylhydrazone.     

Phospholipase D from Soybean (Glycine max L.) Suspension-Cultured Cells: Purification, Structural and Enzymatic Properties

Phospholipase D (phosphatidylcholine phosphatidohydrolase EC3.1.4.4 [EC] ) from soybean (Glycine max L.) suspension-cultured cellwas purified around 1,200-fold to homogeneity by acetone precipitation,Macro-Prep High Q anion exchange, and octyl-Sepharose CL-4Baffinity chromatography. The purified enzyme released 1,600µmol of choline per min per mg of protein. The enzymeis monomeric with a molecular mass of 92 kDa, as estimated bySDS-PAGE. One of the most interesting characteristics of thepurified soybean phospholipase D was the dependence of the pHoptimum on the Ca²⁺ ion concentration in the assay. With 10mM, 20 mM and 40 mM Ca²⁺ ions, the optima were at pH 7.5, 6and 5.5, respectively. The specific adsorption of phospholipaseD onto octyl-Sepharose gel suggests that the molecule becomesmore hydrophobic in the presence of Ca²⁺ ions. The amino acidsequence of the first 18 N-terminal residues of soybean phospholipaseD revealed a high degree of homology with those previously publishedfor cabbage leaf and castor bean endosperm enzymes. Westernblots of the soybean phospholipase D showed an immunoreactivitywith antibodies raised against a synthetic peptide correspondingto the 15 N-terminal aminoacid residues of phospholipase D fromcabbage leaves. (Received March 13, 1995; Accepted May 29, 1995)