Molecular analysis of the genomic organization of the Hymenoptera Diadromus pulchellus and Eupelmus vuilleti

The genomic organization of two parasitic wasps was analyzed by DNA reassociation. Cot curves revealed a pattern with three types of components. A highly repetitive DNA, accounting for 15 to 25% of the genome, was identified as satellite DNA. The moderately repetitive DNA corresponds to 26 to 42% of the genome in both species, and shows large variations in complexity, repetitive frequency and a number of sub-components between males and females. These variations are seen as resulting from DNA amplification during somatic and sexual differentiation. Dot blot analyses show that such DNA amplifications concern several types of structural and regulatory genes. The presence of repeated mobile elements was studied by the Roninson method to compare the repeated sequence patterns of Diadromus pulchellus and Eupelmus vuilleti with those of Drosophila melanogaster. The occurrence and organization of mobile elements in these Hymenoptera differ from those of the neighboring order of Diptera. The repetitive and unique components define very large genomes (1 to 3 × 10⁹ base pairs). The genomic organization in Parasitica appears to be an extreme drosophilan type. We propose that the germinal genome of these parasitic wasps is primarily composed of satellite DNA blocks and very long stretches of unique sequences, separated by a few repeated and/or variously deleted, interspersed elements of each mobile element family.     

Mutation of asparagine 111 of rubisco from Rhodospirillum rubrum alters the carboxylase/oxygenase specificity.

The conserved asparagine 111 of ribulose-1,5-bisphosphate carboxylase/oxygenase from the photosynthetic bacteria Rhodospirillum rubrum was identified as a candidate for a side-chain that might be involved in the carboxylase/oxygenase specificity. It was replaced by site-directed mutagenesis with aspartic acid, leucine, glutamine or glycine residues. The mutant enzymes exhibit a very low carboxylase activity compared with the wild-type enzyme. The values of Km(RuBP) and kcat for Asn111----Gly, the most active mutant, are 420 microM and 0.034 s-1, compared with 13 microM and 3.0 s-1 for wild-type. The mutation of Asn111----Gly causes a more than tenfold decrease in the CO2/O2 specificity factor, tau, tau Asn111----Gly = 0.56 and tau wild-type = 6.7. This is the first reported change in rubisco specificity by a single site-directed mutation alone and suggests a target for future protein engineering studies.     

Nitric oxide causes ADP-ribosylation and inhibition of glyceraldehyde-3-phosphate dehydrogenase.

Nitric oxide and nitric oxide-generating agents like 3-morpholinosydnonimine (SIN-1) stimulate the mono-ADP-ribosylation of a cytosolic, 39-kDa protein in various tissues. This protein was purified from human platelet cytosol by conventional and fast protein liquid chromatography techniques. N-terminal sequence analysis identified the isolated protein as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Nitric oxide stimulates the auto-ADP-ribosylation of GAPDH in a time and concentration-dependent manner with maximal effects after about 60 min. Associated with ADP-ribosylation is a loss of enzymatic activity. NAD(+)-free enzyme is not inhibited by SIN-1, indicating the absolute requirement of NAD+ as the substrate of the ADP-ribosylation reaction. Inhibition of the glycolytic enzyme GAPDH may be relevant as a cytotoxic effect of NO complementary to its inhibitory actions on iron-sulfur enzymes like aconitase and electron transport proteins of the respiratory chain.     

The activation process of Arabidopsis thaliana A1 gene encoding the translation elongation factor EF-1α is conserved among angiosperms

In Arabidopsis thaliana, the activation process of the A1 EF-1 gene depends on several elements. Using the GUS reporter gene, transient expression experiments have shown that mutations of upstream cis-acting elements of the A1 promoter, or the deletion of an intron located within the 5 non-coding region, similarly affect expression in dicot or monocot protoplasts. The results reported here strongly suggest that this 5 intron is properly spliced in Zea mays. We show that two trans-acting factors, specifically interacting with an upstream activating sequence (the TEF 1 box), are present in nuclear extracts prepared from A. thaliana, Brassica rapa, Nicotiana tabacum and Z. mays. In addition, a DNA sequence homologous to the TEF 1 box, found at approximately the same location within a Lycopersicon esculentum EF-1 promoter, interacts with the same trans-acting factors. Homologies found between the A. thaliana and L. esculentum TEF 1 box sequences have allowed us to define mutations of this upstream element which affect the interaction with the corresponding trans-acting factors. These results support the notion that the activation processes of A. thaliana EF-1 genes have been conserved among angiosperms and provide interesting data on the functional structure of the TEF 1 box.     

Theoretical study of ethidium intercalation in triple-stranded DNA and at triplex-duplex junctions.

The contribution of different factors in the interaction of ethidium intercalated into various sequences of a triple helix, or in the region of the junction between the double- and triple-stranded DNA has been studied by energy minimization. It is found that in the total energy of the ethidium- triple helix complexes, a particular electrostatic contribution emerges due to the presence of protonated cytosines in the triple helix. This parameters is determinant in the sequence-specificity of ethidium binding to the triple helix. The preferred intercalation sites of ethidium in the triple helix are proposed. The interaction of ethidium at the triplex-duplex junction, and its effects are also discussed. This study is aimed at searching for new drugs specific for the triple helix, or for the triplex-duplex junctions.     

Extension of the range of recognition sequences for triple helix formation by oligonucleotides containing guanines and thymines.

Oligodeoxynucleotides containing G and T can bind to homopurine.homopyrimidine sequences on double-stranded DNA by forming C.G x G and T.A x T base triplets. The orientation of the third strand in such triple helices depends on the number of GpT and TpG steps. Therefore a single oligonucleotide can be designed to bind to two consecutive homopurine.homopyrimidine sequences where the two homopurine stretches alternate on the two strands of DNA. The oligonucleotide switches from one homopurine strand to the other at the junction between the two sequences. This result shows that it is possible to extend the range of DNA sequences that can be recognized by a single oligonucleotide.     

Evidence for sedative effects of low doses of morphine in mice involving receptors insensitive to naloxone

Low doses of morphine (0.30–2.5 mg/kg) decrease in a dose-dependent manner spontaneous climbing behaviour in mice. This effect is not modified by administration of naloxone at doses up to 1.25 mg/kg. These morphine doses do not modify the locomotor activity but, when they are associated with naloxone (0.5 mg/kg), an obvious inhibition occurs. In rats, a hyperactivity follows the akinesia produced by a morphine administration (10 mg/kg). This hyperactivity is changed into a significant hypokinesia when the animals are treated with naloxone (0.05 mg/kg). These results might reveal a dual effect of low doses of morphine, the excitatory effect of morphine being antagonized by naloxone whereas no action on the sedative effect is observed.