Simultaneous expression of early and late histone messenger RNAs in individual cells during development of the sea urchin embryo

The transition from early (E) to late (L) histone gene expression in developing sea urchin (Strongylocentrotus purpuratus) embryos was examined for H2B, H3, and H4 mRNAs by in situ hybridization of class-specific probes. Hybridization patterns indicate that the shift from E to L mRNAs occurs gradually and simultaneously in all blastomeres. Thus, during the transition the ratio of L to E mRNAs is similar in most cells. This suggests that no sudden changes in histone composition occur in individual cells which might be related to alterations in gene expression associated with differentiation of cell lineages. Around the midpoint of the transition, clusters of cells progressively appear which contain little, if any, E or L histone mRNA. This modulation of expression is coordinated for the three late genes examined because most individual cells contain either high or low levels of all three mRNAs. At blastula stage these clusters of unlabeled cells appear to be randomly distributed throughout the embryo. Subsequently the unlabeled regions expand and are found predominantly in aboral ectoderm as these cells cease to divide. Thus, the L/E histone mRNA ratio is not differentially regulated in diverse cell lineages, and the major differences in total histone mRNA content among individual cells may be related to cell cycle and/or the cessation of division.     

Improved methods for the formation and stabilization of R-loops

Improved methods for the formation and stabilization of R-loops for visualization in the electron microscope are presented. The two complementary strands of a duplex DNA are photochemically crosslinked once every 1 to 3 kb using 4, 5', 8 trimethylpsoralen. R-loops are then formed by incubation with RNA in 70% formamide at a temperature above the DNA melting temperature. Finally, the R-loops are stabilized by modifying the free single strand of DNA with glyoxal, thus minimizing the displacement of the hybridized RNA by branch migration. In this manner R-loops can be formed and visualized at a high frequency irrespective of the base composition of the nucleic acid of interest.     

Nucleotide sequences of the sfuA, sfuB, and sfuC genes of Serratia marcescens suggest a periplasmic-binding-protein-dependent iron transport mechanism.

The cloned sfu region of the Serratia marcescens chromosome confers the ability to grow on iron-limited media to an Escherichia coli K-12 strain that is unable to synthesize a siderophore. This DNA fragment was sequenced and found to contain three genes termed sfuA, sfuB, and sfuC, arranged and transcribed in that order. The sfuA gene encoded a periplasmic polypeptide with calculated molecular weights of 36,154 for the precursor and 33,490 for the mature protein. The sfuB gene product was a very hydrophobic protein with a molecular weight of 56,589. The sfuC gene was found to encode a rather polar but membrane-bound protein with a molecular weight of 36,671 which exhibited strong homology to consensus sequences of nucleotide-binding proteins. The number, structural characteristics, and locations of the SfuABC proteins were typical of a periplasmic-binding-protein-dependent transport mechanism. How Fe3+ is solubilized and taken up across the outer membrane remains an enigma.     

New gas chromatographic-mass spectrometric method for the determination of urinary pyrethroid metabolites in environmental medicine

We have developed and validated a new, reliable and very sensitive method for the determination of the urinary metabolites of the most common pyrethroids in one analytical run. After acidic hydrolysis for the cleavage of conjugates, the analytes cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl(2)CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (trans-Cl(2)CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br(2)CA), 4-fluoro-3-phenoxybenzoic acid (F-PBA) and 3-phenoxybenzoic acid (3-PBA) were extracted from the matrix with a liquid-liquid extraction procedure using n-hexane under acidic conditions. For further clean-up, NaOH was added to the organic phase and the carboxylic acids were re-extracted into the aqueous phase. After acidification and extraction into n-hexane again, the metabolites were then derivatised to volatile esters using N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamid (MTBSTFA). Separation and detection were carried out using capillary gas chromatography with mass-selective detection (GC-MS). 2-Phenoxybenzoic acid (2-PBA) served as internal standard for the quantification of the pyrethroid metabolites. The limit of detection for all analytes was 0.05 microg/l urine. The RSD of the within-series imprecision was between 2.0 and 5.4% at a spiked concentration of 0.4 microg/l and the relative recovery was between 79.3 and 93.4%, depending on the analyte. This method was used for the analysis of urine samples of 46 persons from the general population without known exposure to pyrethroids. The metabolites cis-Cl(2)CA, trans-Cl(2)CA and 3-PBA could be found in 52, 72 and 70% of all samples with median values of 0.06, 0.11 and 0.16 microg/l, respectively. Br(2)CA and F-PBA could also be detected in 13 and 4% of the urine samples.     

An electron microscope study of the relative positions of the 4S and ribosomal RNA genes in HeLa cell mitochondrial DNA

The 4S RNA genes in HeLa mitochondrial DNA (mtDNA) have been mapped by electron microscopy using the electron-opaque label ferritin. This method is based on the high affinity interaction between the protein, avidin, and biotin. 4S RNA, covalently coupled to biotin, was hybridized to single-stranded mtDNA. The hybrids were then labeled with ferritin-avidin conjugates. The positions of ferritin-labeled 4S RNA genes were determined relative to the rRNA genes on both heavy (H) and light (L) strands of mtDNA. This region was recognized as a duplex segment after hybridization either with rRNA in the case of H strands or with DNA complementary to rRNA in the case of L strands.Our studies suggest that at least nineteen 4S RNA genes are present in the HeLa mitochondrial genome. On the H strand, we have confirmed the nine map positions found in a previous electron microscope mapping study (Wu et al., 1972) and obtained evidence for three additional 4S RNA genes. On the L strand, seven 4S RNA genes have been mapped. The nineteen genes are distributed more or less uniformly around the genome. There is a pair of closely spaced genes, approximately 150 nucleotides apart, on the H strand, and another closely spaced pair on the L strand.     

The evolution of nervous system patterning: insights from sea urchin development

Recent studies of the sea urchin embryo have elucidated the mechanisms that localize and pattern its nervous system. These studies have revealed the presence of two overlapping regions of neurogenic potential at the beginning of embryogenesis, each of which becomes progressively restricted by separate, yet linked, signals, including Wnt and subsequently Nodal and BMP. These signals act to specify and localize the embryonic neural fields - the anterior neuroectoderm and the more posterior ciliary band neuroectoderm - during development. Here, we review these conserved nervous system patterning signals and consider how the relationships between them might have changed during deuterostome evolution.     

Space radiation research in Europe: flight experiments and ground-based studies

Exposure to space radiation has long been acknowledged as a potential showstopper for long-duration manned interplanetary missions. In an effort to gain more information on space radiation risk and to develop countermeasures, NASA initiated several years ago a Space Radiation Health Program, which is currently supporting biological experiments performed at the Brookhaven National Laboratory. Accelerator-based radiobiology research in the field of space radiation research is also under way in Russia and Japan. The European Space Agency (ESA) supports research in the field in three main directions: spaceflight experiments on the International Space Station; modeling and simulations of the space radiation environment and transport; and, recently, ground-based radiobiology experiments exploiting the high-energy SIS18 synchrotron at GSI in Germany (IBER program). Several experiments are currently under way within IBER, and so far, beams of C and Fe-ions at energies between 11 and 1,000 MeV/n have been used in cell and tissue targets.