Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria.

We constructed the broad-host-range plasmid pUCD800 containing the sacB gene of Bacillus subtilis for use in the positive selection and isolation of insertion sequence (IS) elements in gram-negative bacteria. Cells containing pUCD800 do not grow on medium containing 5% sucrose unless the sacB gene is inactivated. By using pUCD800, we isolated a 1.4-kilobase putative IS element from Agrobacterium tumefaciens NT1RE by selection for growth on sucrose medium. This putative IS element appears to be unique to Agrobacterium strains.     

In vivo and in vitro studies on the segregation of autonomic and sensory cell lineages

Analysis of interspecific quail/chick chimaeras (made by grafting neural primordium from one species to the other) has demonstrated that the neural crest cell population, which gives rises to a large number of derivatives, including the great majority of peripheral ganglion cells, is pluripotential. When peripheral ganglia themselves are transplanted, it can be shown that many of the developmental potentialities of the parent structure are retained, their ultimate expression depending on the microenvironment in which they become located. One of the conclusions obtained from these in vivo studies, that sensory ganglia contain dormant precursors with autonomic potentialities, has been confirmed and extended by the results of in vitro investigations with dissociated 9- to 15-day embryonic quail dorsal root ganglia. Undetectable during normal embryonic development, adrenergic properties (tyrosine hydroxylase immunoreactivity, radio- and cytochemically demonstrable catecholamine production) develop in a population of small, multipolar cells after four days in culture. This differentiation is strongly dependent on the presence of chick embryo extract in the medium. Unlike the postmitotic primary sensory neurons of the ganglia, many of the adrenergic cells were found to incorporate 3H-thymidine during the culture period. These results support the contention that the latent autonomic percursors belong to the non-neuronal compartment of sensory ganglia.     

Biosynthesis of riboflavin in Bacillus subtilis: origin of the four-carbon moiety.

We studied the incorporation of [1-13C]ribose and [1,3-13C2]glycerol into the riboflavin precursor 6,7-dimethyl-8-ribityllumazine, using a riboflavin-deficient mutant of Bacillus subtilis. The formation of the pyrazine ring requires the addition of a four-carbon moiety to a pyrimidine precursor. The results show that C-6 alpha, C-6, C-7, and C-7 alpha of 6,7-dimethyl-8-ribityllumazine were biosynthetically equivalent to C-1, C-2, C-3, and C-5 of a pentose phosphate. C-4 of the pentose precursor was lost through an intramolecular skeletal rearrangement. Thus, the last steps in the biosynthesis of 6,7-dimethyl-8-ribityllumazine apparently involve the same mechanism in bacteria as in fungi.     

Thermography as a predictive tool for laser treatment of port-wine stains

The argon laser, which has been proven both useful and safe for port-wine stain therapy, interacts with the hemoglobin of the vessels. In a percentage of cases, this treatment is still inefficient, and there is a lack of correlation between these bad results and clinical or histologic criteria. Thermography, which explores the vascularization of the port-wine stain, leads us to consider port-wine stains from a physical point of view. This very simple test shows no correlation with the clinical parameters of port-wine stain but is closely related to the results obtained with laser therapy. It seems to be a good criterion to estimate the argon laser treatment prognosis.     

Structure of mouse interferon-beta cDNA clones: sequence rearrangements in a cloned cDNA

A fraction enriched in interferon (IFN) mRNA was prepared from mouse C243-3 induced cells and was used for the construction of a cDNA library. Two plasmids were obtained after screening by differential colony hybridization and IFN mRNA hybridization-selection and translation. The nucleotide sequences of the cDNA inserts revealed that both were partial copies of IFN-beta mRNA. The cDNA 861 corresponds to the entire 3' nontranslated region of the mRNA while the cDNA 2939 consists of rearranged translated regions of IFN mRNA. A mechanism for the rearrangement events during cDNA synthesis is proposed. A chromosomal DNA fragment hybridizing to cDNA 2939 was identified by screening a mouse genomic library.     

Synthesis and phosphorylation of cytoskeleton components in foetal,regenerating and adult normal rat hepatocytes during culture

Summary Detergent insoluble material (DIM) was prepared by gentle treatment with detergent from foetal, regenerating and adult normal rat hepatocytes cultured for various times. It retained to some degree the morphology of the cells. After incubation of intact cells with ³⁵S-methionine, most of the labelled DIM proteins were found to be components of the cytoskeleton. They included several cytokeratins, vimentin and actin. The synthesis rate varied with the age of animals and culture conditions. The high synthetic rate of vimentin in foetal and regenerating hepatocytes could be associated with cell proliferation. No correlation was found between cytokeratin synthesis and hepatocyte growth. Most of the cytoskeleton proteins could be phosphorylated in intact cells and in DIM from cultured hepatocytes. However the degree of phosphorylation of these proteins was not related to their synthetic rate. The decreased phosphorylation level in cultured adult rat hepatocytes could be related to the rapid loss of specific functions.     

Effect of B-Z transition and nucleic acid structure on the conformational dynamics of bound ethidium dimer measured by hydrogen deuterium exchange kinetics.

Ethidium dimer is shown to bind by intercalation, almost equally well, to the B and Z form of poly[(dG-m5dC)].poly[(dG-m5dC)], whereas the ethidium monomer shows a strong preference for the B form. The hydrogen-deuterium (H-D) exchange kinetics of the ethidium dimer bound to the B and Z form of poly [(dG-m5dC)].poly[(dG-m5dC)] could then be compared. The kinetics of the H-D exchange were strikingly slower when the dye was bound to Z DNA as compared to B DNA. The exchange kinetics were also modified when ethidium dimer was bound to tRNA and to a triple stranded structure. It is proposed that a dynamic fluctuation at the level of the nucleic acid could modulate the dynamic fluctuation at the level of the bound ligand.     

Unusual sialilation of three different rare genetic variants of serum DBP: Gc 1A17, Gc 1A16, and Gc 1A11

Summary The proteins of three anodal Gc1 variants, Gc 1A16, 1A11, and 1A17, are characterized by the most acidic isoelectric points observed so far among the different Gc mutants. Stepwise removal of N-acetylneuraminic acid (NANA) by treatment with neuraminidase was performed to estimate the degree of sialilation of these Gc variants. The results indicate that both proteins, the anodal and the cathodal component of these Gc 1 mutants, carry sialic acid residues. This observation is remarkable in so far as usually only the anodal component of the Gc 1 protein contains NANA and only a single residue. From the experiments carried out it can be deduced that Gc 1A16 has two NANA residues in the anodal and one NANA residue in the cathodal component. Gc 1A16 was found in four members of three generations in a Danish family; the variant segregated as a Mendelian trait. More difficult to interprete are the results obtained with the variants Gc 1A11 and Gc 1A17. Gc 1A11 probably has three NANA residues in the anodal and two NANA residues in the cathodal component. Gc 1A11 has been observed in two mother-child pairs and is presumably also a simple genetic trait. Gc 1A17 has also several NANA residues in both Gc proteins; it is suggested that the anodal component has either three or four NANA residues and the cathodal component either two or three NANA residues. Family information on this variant is not yet available.     

Expression of the adrenergic phenotype by dorsal root ganglion cells of the quail in culture in vitro

From the results of previous studies, we have suggested that "autonomic" cell precursors exist in latent form in sensory ganglia of avian embryos. The potentialities can be expressed when the ganglia are transplanted into a young embryo host. In the present study, we have observed a similar transformation in cultures of dissociated dorsal root ganglia taken from quail embryos of 7-15 days of incubation. From the 4th day of culture onward, numerous adrenergic cells appear. They display tyrosine hydroxylase immunoreactivity, synthesise and store catecholamines and generally differ in size and shape from primary sensory neurons. They and/or their precursors can actively proliferate in culture. The differentiation of these catecholaminergic cells, which can not be detected in quail dorsal root ganglia during normal development in vivo, is dependent on one or more factors present in 9-day chick embryo extract.