Molecular genetic analysis of an FNR-dependent anaerobically inducible Escherichia coli promoter

From the effects of 13 deletions and three linker-scanner mutations at the Escherichia coli nirB promoter we have located sequences necessary for FNR-dependent induction of activity by anaerobiosis and further nitrite-dependent stimulation of expression. We describe a nirB promoter derivative that allows the cloning of 'cassettes' carrying different FNR-binding sequences and experiments in which a number of point mutations were introduced into these sequences. FNR-dependent stimulation of expression from the nirB promoter is critically dependent on the location of the FNR-binding site, and deletion or insertion of one base pair is sufficient to disrupt promoter function. We have transferred a number of cassette FNR-binding sequences from the nirB promoter to the unrelated melR promoter. The insertion of FNR-binding sequences at the melR promoter is sufficient to confer fnr-dependency on expression. However expression from these hybrid promoters is not as efficiently repressed during aerobic growth, suggesting that the function of bound FNR is dependent on the sequence context of the FNR-binding sequence.     

Selenocysteine's mechanism of incorporation and evolution revealed in cDNAs of three glutathione peroxidases

The nonsense codon, UGA, has for the first time recently been shown to encode selenocysteine in two proteins, mouse glutathione peroxidase (GSH-Px) (EC 1.11.1.9) and bacterial formate dehydrogenase. A co-translational rather than post-translational selenium-incorporation mechanism has been implicated. Furthermore, high expression levels of GSH-Px have suggested that suppression of termination is efficient and specific. We have isolated and characterized pituitary, kidney and placenta cDNAs for bovine, human and mouse GSH-Px respectively. It is demonstrated that this novel suppression event occurs in diverse tissues, in at least three mammalian species and at the translational step. Surprisingly, GSH-Px is shown to be extramitochondrially encoded, indicating a cytosolic suppression event rather than one utilizing the mitochondria's well-documented extended codon-reading ability. Sequence analysis reveals that a simple proximal contextual pattern responsible for readthrough does not exist. Analysis of predicted secondary structures of mRNAs, however, has revealed a conformation which may be unique to selenocysteine proteins and may prove useful as a tool for artificial incorporation of selenocysteines. A human intron for GSH-Px from an unspliced mRNA has been isolated whose position indicates an ancient, divergent evolutionary relationship with thioredoxin-S2, rather than an independent convergent one.     

Induction of transforming growth factor-alpha in activated human alveolar macrophages

The early monocyte infiltration observed in normal wound repair and in a number of pathologic processes precedes the epithelial and connective tissue proliferative responses, suggesting that the monocyte/macrophage may be an important source of growth factors for these tissues. In culture, activated macrophages secrete growth factors active on fibroblasts, smooth muscle, endothelium, and epithelium. This report demonstrates that activated human alveolar macrophages express the gene for transforming growth factor-alpha (TGF-alpha) in an inducible manner and secrete a factor into the culture medium that is functionally and immunologically identical to TGF-alpha. Two different molecular species of TGF-alpha activity (approximately 8,500-12,000 and 28,500 daltons) are identified in macrophage-conditioned medium. These observations establish the macrophage as a diploid human cell capable of synthesizing and secreting TGF-alpha. The activated macrophage therefore represents a cellular source of a mitogenic factor that is potentially important in epithelial proliferation and repair.     

Characterization and possible opioid modulation of N-methyl-D-aspartic acid induced increases in serum luteinizing hormone levels in the developing male rat

It has been previously reported that the excitatory amino acid, N-methyl-D-aspartic acid (NMDA), elicits prompt increases in serum luteinizing hormone (LH) levels in young male rats. The present studies were carried out to determine whether the effects of NMDA on LH were mediated by the release of LHRH from the hypothalamus. We also examined whether NMDA-sensitive neuronal pathways interacted with the endogenous opioid system regulating LHRH release and the ontogeny of NMDA-evoked increases in serum LH. We found that the age-response curve for NMDA-induced increases in LH was an inverted U; at early ages (10 and 15 days) the amino acid was marginally effective in increasing LH levels, it became maximally effective from post-natal days 20-40 and thereafter rapidly lost its efficacy such that it was virtually inactive in adult animals. Dose-response curves revealed that adult animals were more than 10-fold less sensitive to NMDA than their younger counterparts. Our studies also demonstrated that NMDA increased LH via a direct effect on the hypothalamic release of LHRH since a potent LHRH antagonist competitively inhibited the effects of NMDA. Finally, we observed that morphine competitively inhibited the effects of NMDA on LH release, suggesting a relationship between NMDA-sensitive neuronal pathways and those endogenous opioid-containing systems which are known to regulate LH release.     

Suppressible P-element alleles of the vestigial locus in Drosophila melanogaster

Summary A series of P-element insertion mutations at one site in the vestigial (vg) locus was tested for cytotype dependent effects on vg expression. The mutant phenotypes for four P-element vg alleles were suppressed when the alleles were stabilized in the P-cytotype. The suppression was observed whenever repressor-producing P-elements were present in the genome. Genetic and molecular analysis indicated that the suppression is not due to excision or other irreversible alterations of the inserts. The results are consistent with a model in which somatic P-element repressor binding to the ends of P-element inserts can modify the effects of these inserts on target gene expression.     

Decreased incidence of mycorrhizal root tips associated with soil heavy-metal enrichment

Mycorrhizal incidence was studied at two forested locations in south-central Virgina. At each location, one site with soil naturally enriched in copper, lead and zinc was designated as mineralized, and an adjacent site, with significantly lower levels of these metals, was used as a control. A total of 223 soil samples were collected during the summer of 1984 and assayed for active mycorrhizal tips. A reduced active mycorrhizal root tip count was found in those samples collected from the mineralized sites at both locations (P≤0.001).     

Purification of proteins from polyacrylamide gels, free of detergent or dye

A two step procedure recovers proteins from sodium dodecyl sulfate polyacrylamide gels. The proteins are eluted by electrophoretic dialysis. The eluent is then passed through an Amberlite CG-400 anion-exchange resin. The recovery of protein is nearly total. The recovered proteins have no detectable sodium dodecyl sulfate contamination. With gels that have been stained with Coomassie Brilliant Blue R, the procedure recovers the proteins free of the dye. We have used this procedure successfully during the purification of epidermal glycoproteins.