Methylation and expression of a metallothionein promoter ovine growth hormone fusion gene (MToGH1) in transgenic mice

We have examined transgene methylation in the DNA from the livers of a pedigree of mice carrying three copies of an integrated MToGH1 transgene. Utilizing the methylation-sensitive isoschizomersMsp I andHpa II, Southern blot analysis revealed that all second generation animals derived from a transgenic female had hypermethylated DNA, whereas first generation animals sired by a transgenic male displayed a range of methylation phenotypes ranging from no methylation to hypermethylation of the transgene sequences. Of the mice that exhibited hypermethylation of the transgene in CpG dinucleotides (CmCGG), a minority of these animals also exhibited apparent CpC methylation (i.e. inhibition ofMsp I cutting, presumably blocked by methylation of the outer C of CCGG). Methylation was also examined in the inner C of CC(A/T)GG sequences in the MToGH1 transgene using the isoschizomer pairBstN I andEcoR II. A minority of MToGH1 animals in the F₁ generation showed clear evidence of methylation in these sites as well as in the inner and outer Cs of CCGG sites. An examination of MToGH1 expression in terms of oGH levels in serum revealed that there was a high degree of variation in the levels of circulating oGH between animals of this pedigree. There was a weak inverse relationship between the serum level of oGH and the extent of methylation of the transgene. In particular, mice exhibiting CpC together with CpG methylation were found to have very low levels of circulating oGH. Our results highlight the nature and complexity of epigenetic factors associated with transgene sequences which may ultimately influence expression of introduced genes in the mammalian genome.     

Biogenesis of mitochondria: Defective yeast H+-ATPase assembled in the absence of mitochondrial protein synthesis is membrane associated

We have investigated the extent to which the assembly of the cytoplasmically synthesized subunits of the H⁺-ATPase can proceed in a mtDNA-less (rho°) strain of yeast, which is not capable of mitochondrial protein synthesis. Three of the membrane sector proteins of the yeast H⁺-ATPase are synthesized in the mitochondria, and it is important to determine whether the presence of these subunits is essential for the assembly of the imported subunits to the inner mitochondrial membrane. A monoclonal antibody against the cytoplasmically synthesized -subunit of the H⁺-ATPase was used to immunoprecipitate the assembled subunits of the enzyme complex. Our results indicate that the imported subunits of the H⁺-ATPase can be assembled in this mutant, into a defective complex which could be shown to be associated with the mitochondrial membrane by the analysis of the Arrhenius kinetics of the mutant mitochondrial ATPase activity.This paper is No. 61 in the seriesBiogenesis of Mitochondria. For paper No. 60, see Novitskiet al. (1984).     

In vitro autoradiographic localization of vasoactive intestinal peptide (VIP) binding sites in the rat central nervous system

This paper describes the autoradiographic distribution of VIP binding sites in the rat central nervous system using monoiodinated ¹²³I-labeled VIP. High densities of VIP binding sites are observed in the granular layer of the dorsal dentate gyrus of the hippocampus, the basolateral amygdaloid nucleus, the dorsolateral and median geniculate nuclei of the thalamus as well as in the ventral part of the hypothalamic dorsomedial nucleus.     

Gecko phonotaxis to cricket calling song: A case of satellite predation

Insectivorous Mediterranean house geckos, Hemidactylus tursicus, are found in the field close to the burrows of calling male decorated crickets, Gryllodes supplicans. In playback experiments where adult geckos were presented with calls of these crickets or calls of frogs as controls, geckos exhibited positive phonotaxis to broadcast cricket calling song. This indicates that geckos orient to and approach male cricket calls, even though the crickets call from burrows where they are protected from the geckos. However, this behaviour enables the geckos to intercept and consume female crickets that also respond phonotactically to the cricket calls. Thus geckos act as ‘satellite predators’, a situation which may impose sex-biased mortality on female crickets.     

Calmodulin during development and metamorphosis in urodelan amphibians

Calmodulin isolated and purified to homogeneity from young larvae is very similar to that obtained from adult Pleurodeles waltlii and these proteins are almost identical to previously described vertebrate calmodulins. During P. waltlii development, an increase in total individual calmodulin content is observed after the heart beating stage. In dorsal axial muscle, calmodulin level which is very high at the beginning of larval life (premetamorphosis) decreases strikingly in the first part of prometamorphosis. Such an evolution is observed in Ambystoma mexicanum too. Then, a significant increase occurs during metamorphosis. In contrast, calmodulin level in P. waltlii cardiac ventricular muscle increases continuously from hatching to the end of metamorphic climax. Thyroxine treatment which promotes precocious metamorphosis in P. waltlii and experimental metamorphosis in neotenic A. mexicanum, induces a rapid and significant increase in muscle calmodulin concentration.     

What's new in lysozyme research?

The present review is focused on the main achievements realized in the lysozyme research field since the meeting held in 1972 to commemorate the fiftieth anniversary of the discovery of this enzyme. Despite of extensive structural, physico-chemical, crystallographic, genetic, immunological and evolutionary studies devoted to lysozymes, their biological role is still not exactly known.     

A sensitive fluorimetric microassay for the determination of glutathione peroxidase activity. Application to human blood platelets

The method proposed for measuring glutathione peroxydase (GSH-Px) activity is based on the determination of oxidized glutathione (GSSG) using o-phtalaldehyde (OPT) as a fluorescent reagent. This method makes it possible to study the kinetics of both substrates (peroxide and reduced glutathione, GSH), and allosteric kinetics were found for GSH, with human platelets as the source of GSH-Px. Different methods for platelet disruption were compared. The reference values obtained for GSH-Px activity in human blood platelets by this fluorimetric procedure and the conventional enzymatic method were very similar and significantly higher than those previously reported; the reasons for this difference are discussed.     

The use of specific lysine modifications to locate the reaction site of cytochrome c with sulfite oxidase

The reduction of cytochrome c by beef liver sulfite oxidase was found to be strongly inhibited by high ionic strength, indicating the importance of electrostatic interactions to the reaction. The reaction rates of sulfite oxidase with singly trifluoroacetylated or trifluoromethylphenylcarbamylated cytochrome c derivatives were studied to determine the role of individual lysines in the reaction. The reaction rate was decreased by modification of the lysines immediately surrounding the heme crevice, the decreases following the order: Lys 13 > Lys 25 Lys 79 ≈ Lys 87 > Lys 8 ≈ Lys 27 ≈ Lys 72. Modification of lysines 22, 55, 88, 99, and 100 had no effect on the reaction rate. These results indicate that the interaction site on cytochrome c for sulfite oxidase is at the heme crevice region, and overlaps considerable with that for cytochrome oxidase.