Identification and purification of overlapping cosmid clones of the region Xq24-qter of human X chromosome using HPLC.

The assembly of a large physical map of genomes requires simultaneous analysis of many cosmid clones for overlapping regions. The search for overlapping regions may be achieved by various means. High-performance liquid chromatography (HPLC) provides an alternative to gel electrophoresis since microgram amounts of each DNA fragment may be collected into individual test tubes for further analysis. HPLC has been used to identify overlapping cosmid clones from a pool of cosmid DNA containing the terminal portion of the long arm of the human X chromosome (Xq24-qter). Among 400 cosmids analyzed, 3 were shown to overlap.     

Tyrosination-detyrosination of tubulin and microtubules during the development of chick erythrocytes

The protein composition of nuclear matrices containing different amount of DNA was examined. It was found that, in matrices containing 2% to 80% of total DNA, the quantity of DNA-bound proteins remains relatively constant varying from 10% to 15% of total nuclear proteins. Electrophoretic patterns do not differ substantially, but autoradiograms with in vitro ¹²⁵I labelled proteins show quantitative variations in the actin content. Application of radioimmunoassay (RIA) enabled to determine the exact content of actin in GAT nuclei and nuclear matrices – 5 g/ml in nuclei, of which 50% are bound to DNA and 3001o being a component of the protein part of the nuclear matrix. These results are supported by electron microscopic data, where immunogold technique was performed on thin sections and spread material. The applied methods suggest that part of the nuclear actin is tightly bound (resistant to 2 M NaCI) to DNA and represents a component of the internal nuclear matrix.     

Actions of Excitatory Amino Acids on Somatostatin Release from Cortical Neurons in Primary Cultures

L-Glutamate, N-methyl-D-aspartic acid (NMDA), quisqualate, and kainate were found to increase endogenous somatostatin release from primary cultures of rat cortical neurons in a dose-dependent manner. The rank order of potency calculated from the dose-response curves was quisqualate greater than glutamate = NMDA greater than kainate, with EC50 values of 0.4, 20, and 40 microM, respectively. Alanine, glutamine, and glycine did not modify the release of somatostatin. The stimulation of somatostatin release elicited by L-glutamate was Ca2+ dependent, was decreased by Mg2+, and was blocked by DL-amino-5-phosphonovaleric acid (APV) and thienylphencyclidine (TCP), two specific antagonists of NMDA receptors. The NMDA stimulatory effect was strongly inhibited by APV in a competitive manner (IC50 = 50 microM) and by TCP in a noncompetitive manner (IC50 = 90 nM). The release of somatostatin induced by the excitatory amino acid agonists was not blocked by tetrodotoxin (1 microM), a result suggesting that tetrodotoxin-sensitive, sodium-dependent action potentials are not involved in the effect. Somatostatin release in response to NMDA was potentiated by glycine, but the inhibitory strychnine-sensitive glycine receptor did not appear to be involved. Our data suggest that glutamate exerts its stimulatory action on somatostatin release essentially through an NMDA receptor subtype.     

Phosphotransfer reactions as a means of G protein activation

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) serve to transduce information from agonist-bound receptors to effector enzymes or ion channels. Current models of G protein activation-deactivation indicate that the oligomeric GDP-bound form must undergo release of GDP, bind GTP and undergo subunit dissociation, in order to be in active form (GTP bound subunits and free dimers) and to regulate effectors. The effect of receptor occupation by an agonist is generally accepted to be promotion of guanine nucleotide exchange thus allowing activation of the G protein. Recent studies indicate that transphosphorylation leading to the formation of GTP from GDP and ATP in the close vicinity, or even at the G protein, catalysed by membrane-associated nucleoside diphosphate kinase, may further activate G proteins. This activation is demonstrated by a decreased affinity of G protein-coupled receptors for agonists and an increased response of G protein coupled effectors. In addition, a phosphorylation of G protein subunits and consequent phosphate transfer reaction resulting in G protein activation has also been demonstrated. Finally, endogenously formed GTP was preferentially effective in activating some G proteins compared to exogenous GTR The aim of this report is to present an overview of the evidence to date for a transphosphorylation as a means of G protein activation (see also refs [1 and 2] for reviews). (Mol Cell Biochem 157: 593, 1996)Recipient of Servier Investigator Award     

Mapping of the mouse Tdgf1 gene and Tdgf pseudogenes

Teratocarcinoma-derived growth factor-1 (Tdgf1), a member of the ``EGF family' of growth factors, is expressed during mouse gastrulation in the forming mesoderm and later in the truncus arteriosus of the developing heart. In humans, TDGF1 is highly expressed in germ cell tumors and in colon and mammary carcinomas. In mouse, one gene (Tdgf1) and two pseudogenes (Tdgf1-ps1 and Tdgf1-ps2) have been isolated and characterized. Tdgf1 corresponds to the gene expressed in F9 teratocarcinoma cells. Tdgf1-ps1 and Tdgf1-ps2 are two intronless sequences with all the characteristics of retroposons. In the present paper, we assign the chromosomal location for Tdgf1, Tdgf1-ps1, and Tdgf1-ps2 sequences to Chromosomes (Chrs) 9, 16, and 17, respectively. Two previously described mouse mutants, scant hair (sch) and fur deficient (fd), map near the Tdgf1 gene. Analysis of their DNA coding region provided no evidence that Tdgf1 could be the responsible gene for these phenotypes. Finally, analysis of the DNA from several Mus musculus strains and from Mus spretus mice revealed a highly variable restriction pattern and the absence of the Tdgf1-ps1 genomic sequence from the Mus spretus genome. Received: 23 November 1996 / Accepted: 17 February 1997     

Ultrastructure of the sporophyte foot-gametophyte vaginula complex inTimmiella barbuloides (Brid.) Moenk

The sporophyte foot of the mossTimmiella barbuloides consists of an unistratose epidermis of transfer cells, a parenchymatous cortex, and a small central strand consisting only of hydroids. The parenchymatous tissue of the vaginula develops one layer of transfer cells opposite the foot, whose lower extremity extends into the gametophyte stem's central strand. From the bottom to the top of the foot the ultrastructure of the sporophyte transfer cells shows some gradual changes that appear related to a functional specialization of these cells. According to a centripetal gradient, the quantity of plastid starch progressively lessens in both vaginula parenchyma and foot cortex. the observed morphological patterns suggest that in the foot-vaginula complex nutrients are translocated radially up to the sporophyte central strand.