Light and electron microscopical postembedding lectin histochemistry for WGA-binding sites in the renal cortex of the mouse embedded in polyhydroxy aromatic resins LR-White and LR-Gold

Summary This work describes a technique which permits study of the postembedding lectin histochemistry for WGA-binding sites at the light and electron microscopical level on the same resin embedded tissue without removing or etching of the resin. Unfixed kidney pieces or kidney pieces fixed in 4% formaldehyde were embedded in the hydrophilic polyhydroxy aromatic resins LR-Gold and LR-White, following dehydration in up to 70% ethanol, 90% ethanol or 100% ethanol. LR-Gold was cryopolymerised at –25° C using the light sensitive initiator benzil, whereas LR-White was heat-cured at +50° C. The localisation of WGA-binding sites at the light microscopical level was investigated using FITC-labelled WGA. The ultrastructural localisation of WGA-binding sites was performed using 15 nm gold-labelled WGA. The best fluorescence staining results were obtained on fixed or unfixed tissue dehydrated in up to 70% ethanol and embedded in LR-Gold. At the ultrastructural level, the best staining results for WGA-binding sites were seen on tissue sections, dehydrated in up to 90% ethanol prior to embedding in LR-Gold.     

Appearance of lectin-binding sites during vascularization of the primordium of the central nervous system in 10 to 12-day-old mouse embryos

Summary In the present study lectin-binding sites were investigated for the lectins Ricinus communis agglutinin (RCA I), wheat germ agglutinin (WGA), soya bean agglutinin (SBA), concanavalin A (Con A), Lotus tetragonolobus(LTA) and Limulus polyphemus agglutinin (LPA) during the initial stages of vasculogenesis of the CNS-anlage in 10 to 12-day-old NMRI mouse embryos. Specific binding sites for the lectins RCA I (sugar specificity: -D-galactose, N-acetylgalactosamine), WGA (sugar specificity: N-acetylglucosamine, sialic acid), and SBA (sugar specificity: N-acetylgalactosamine, -D-galactose) were detected in the newly formed capillaries within the neuroepithelial cell layer. In contrast, binding sites for Con A, LTA and LPA could not be observed at the start of the vascularization of the CNS-anlage. From these results, the conclusion can be drawn that glycoconjugates containing D-galactose, N-acetylgalactosamine and N-acetyl-glucosamine moieties are involved in the early vasculogenesis of the embryonic CNS-anlage of the mouse.     

Transgenic mice generated by pronuclear injection of a yeast artificial chromosome.

Transgenic mice have become invaluable for analysing gene function and regulation in vivo. However, the size of constructs injected has been limited by the cloning capacity of conventional vectors, a constraint that could be overcome with yeast artificial chromosomes (YACs). We investigated the feasibility of making transgenic mice with YACs by pronuclear injection of a small YAC carrying a gene encoding tyrosinase. Use of a vector with a conditional centromere allowed fifteenfold amplification of the YAC in yeast and its recovery in high yield. The albino phenotype of the recipient mice was rescued demonstrating the correct expression of the tyrosine gene from the construct. Furthermore, the telomeric sequences added by the yeast integrated into the mouse genome and did not reduce efficiency of integration. Using this technique future experiments with longer YACs will allow the expression of gene complexes such as Hox and the globin gene clusters to be analysed in transgenic animals.     

Physical mapping of two Xp markers DXS16 and DXS143

Summary Lymphocyte karyotyping of an infant girl with the clinical features of microphthalmia, iridoschisis, goiter, hip joint dysplasia, labium synechia and craniotabes revealed an Xp deletion. The lymphocyte karyotypes of the parents were normal. Bromodeoxyuridine incorporation studies showed that, in 42 out of 43 metaphases, the deleted X chromosome was late replicating. In one metaphase, the normal X chromosome was observed to be allocyclic. Using DNA markers from the Xp22 region, the breakpoint was assigned distal to DXS16 (pXUT23) and proximal to DXS143 (dic56). Dosage intensity measurements confirmed that the STS gene and the DNA marker DXS31 were involved in the deleted area. Restriction fragment length polymorphism analysis revealed that the paternally derived X-chromosome was deleted.     

Abortive Infection of F-Plasmid-Containing Escherichia coli Cells by Bacterial Virus T7 Is Determined by the Right End of T7 Gene 1

Phage T7 infects male (F-plasmid-carrying) Escherichia coli cells abortively, whereas the closely related phage T3 grows normally. The inability or ability of phage to replicate in male host cells depends on whether the right end of gene 1 (coding for the phage-specific RNA polymerase) consists of T7 or T3 DNA base sequences.     

Germline integration of moloney murine leukemia virus at the Mov13 locus leads to recessive lethal mutation and early embryonic death

Thirteen mouse substrains genetically transmitting the exogenous Moloney murine leukemia virus (M-MuLV) at a single locus (Mov locus) have been derived previously. Experiments were performed to investigate whether homozygosity at the Mov loci would be compatible with normal development. Animals heterozygous at an Mov locus were mated, and the genotype of the offspring was analyzed. From parents heterozygous at the loci Mov1 to Mov12, respectively, homozygous offspring were obtained with the expected Mendelian frequency. In contrast, no homozygous offspring or embryos older than day 15 of gestation were obtained from parents heterozygous at the Mov13 locus. When pregnant Mov13 females at day 13 and day 14 of gestation were analyzed, approximately 25% of the embryos were degenerated. Genotyping revealed that these degenerated embryos were invariably homozygous and the normal appearing embryos were either heterozygous or negative for M-MuLV. These results suggest that integration of M-MuLV at the Mov13 locus leads to insertion mutagenesis, resulting in embryonic arrest between day 12 and day 13 of gestation. It is possible that the Mov13 locus represents a gene or gene complex involved in the early embryonic development of the mouse.     

Enzymatic methylations: III. Cadaverine-induced conformational changes of E.coli tRNAfMet as evidenced by the availability of a specific adenosine and a specific cytidine residue for methylation+

A partially purified tRNA methylase fraction from rat liver, containing m(2)G- m(1)A- and m(5)C-methylase, was used to study the influence of Mg(++) and of the biogenic polyamine cadaverine on the enzymatic methylation of E.coli tRNA(fMet)in vitro. In presence of 1 or 10 mM Mg(++), guanosine no. 27 was methylated to m(2)G. In 1 mM Mg(++) plus 30 mM cadaverine, guanosine in position 27 and adenosine in position 59 were methylated. In presence of 30 mM cadaverine alone tRNA(fMet) accepted three methyl groups: in addition to guanosine no. 27 and adenosine no. 59 cytidine no. 49 was methylated. In order to correlate tRNA(fMet) tertiary structure changes with the methylation patterns, differentiated melting curves of tRNA(fMet) were measured under the methylation conditions. It was shown that the thermodynamic stability of tRNA(fMet) tertiary structure is different in presence of Mg(++), or Mg(++) plus cadaverine, or cadaverine alone. From the differentiated melting curves and from the methylation experiments one can conclude that at 37 degrees in the presence of Mg(++) tRNA(fMet) has a compact structure with the extra loop and the TpsiC-loop protected by tertiary structure interactions. In Mg(++) plus cadaverine, the TpsiC-loop is available, while the extra loop is yet engaged in teritary structure (G-15: C-49) interactions. In cadaverine alone, the TpsiC-loop and the extra loop are free; hence under these conditions the open tRNA(fMet) clover leaf may be the substrate for methylation. In general, cadaverine destabilizes tRNA tertiary structure in the presence of Mg(++), and stabilizes tRNA(fMet) tertiary structure in the absence of Mg(++). This may be explained by a competition of cadaverine with Mg(++) for specific binding sites on the tRNA. On the basis of these experiments a possible role of biogenic polyamines in vivo may be discussed: as essential components of procaryotic and eucaryotic ribosomes they may together with ribosomal factors facilitate tRNA-ribosome binding during protein biosynthesis by opening the tRNA tertiary structure, thus making the tRNA's TpsiC-loop available for interaction with the complementary sequence of the ribosomal 5S RNA.     

Reduction of cardiac hypertrophy in TGR(mREN2)27 by angiotensin II receptor blockade

TGR(mREN2)27 is a transgenic rat harboring the murine Ren-2 gene and exhibit fulminant hypertension and marked heart hypertrophy. In order to study the role of angiotensin II in the increase of cardiac mass, these animals were treated with anti-hypertensive and non-antihypertensive doses of the angiotensin II receptor AT₁ antagonist Telmisartan for 9 weeks. All doses led to significant reductions of heart hypertrophy detected by the evaluation of the diameter of cardiac muscle bundles. We conclude from this study that cardiac hypertrophy in TGR(mREN2)27 is characterized by an increased volume of cardiomyocytes and an unchanged amount of fibrous tissue and that angiotensin II plays an important role in the mechanisms leading to this phenotype.     

Xenobiotic biotransformation in unicellular green algae. Involvement of cytochrome P450 in the activation and selectivity of the pyridazinone pro-herbicide metflurazon.

The N-demethylation of the pyridazinone pro-herbicide metflurazon into norflurazon implies a toxification in photosynthetic organisms. This is confirmed by quantitative structure activity relationships determined for two unicellular green algae, Chlorella sorokiniana and Chlorella fusca; however, the latter is 25 to 80 times more sensitive to metflurazon. This sensitivity is linked to differences in the N-demethylase activity of both algae, as determined by an optimized in vivo biotransformation assay. Apparent K(m) values of the metflurazon-N-demethylase indicate a 10-fold higher affinity for this xenobiotic substrate for Chlorella fusca. Furthermore, algal metflurazon-N-demethylation is characterized by distinct variations in activity, depending on the stage of cell development within the cell cycle. Several well-established inhibitors of cytochrome P450-mediated reactions, including piperonylbutoxide, 1-aminobenzotriazole, 1-phenoxy-3-(1H-1,2,4-triol-1yl)-4-hydroxy-5,5-dimethylhexane++ +, and tetcyclacis, as well as cinnamic acid, a potential endogenous substrate, inhibited the N-demethylation of metflurazon. The results suggest that the N-demethylation of metflurazon by both algae is mediated by a cytochrome P450 monooxygenase. The determination of antigenic cross-reactivity of algal proteins with heterologous polyclonal antibodies originally raised against plant P450s, anti-cinnamic acid 4-hydroxylase (CYP73A1), anti-ethoxycoumarin-O-dealkylase, anti-tulip allene oxidase (CYP74), and an avocado P450 (CYP71A1) or those of bacterial origin, CYP105A1 and CYP105B1, suggests the presence of distinct P450 isoforms in both algae.     

The possible roles of residues 79 and 80 of the Trp repressor from Escherichia coli K-12 in trp operator recognition

We constructed mutants of the Trp repressor from Escherichia coli K-12 with all possible single amino acid exchanges at positions 79 and 80 (residues 1 and 2 of the recognition helix). We tested these mutants in vivo by measuring the repression of synthesis of β-galactosidase with symmetric variants of α- and β-centered trp operators, which replace the lac operator in a synthetic lac system. The Trp repressor carrying a substitution of isoleucine 79 by lysine, showed a marked specificity change with respect to base pair 7 of the α-centered trp operator. Gel retardation experiments confirmed this result. Trp repressor mutant IR79 specifically recognizes a trp operator variant with substitutions in positions 7 and 8. Another mutant, with glycine in position 79, exhibited loss of contact at base pair 7. We speculate that the side chain of Ile79 interacts with the AT base pairs 7 and 8 of the α-centered trp operator, possibly with the methyl groups of thymines. Replacement of thymine in position 7 or 8 by uracil confirms the involvement of the methyl group of thymine 8 in repressor binding. Several Trp repressor mutants in position 80 (i.e. AI80, AL80, AM80 and AP80) broaden the specificity of the Trp repressor for α-centered trp operator variants with exchanges in positions 3, 4 and 5.