Experimental Indication in Favor of the Introns-Late Theory: The Receptor Tyrosine Kinase Gene from the Sponge Geodia cydonium

We have analyzed the gene that encodes receptor tyrosine kinase (RTK) from the marine sponge Geodia cydonium, which belongs to the most ancient and simple metazoan groups, the Porifera. RTKs are enzymes found only in metazoa. The sponge gene contains two introns in the extracellular part of the protein. However, the rest of the protein (transmembrane and intracellular part), including the tyrosine kinase (TK)-domain, is encoded by a single exon. In contrast, all TK genes, so far known only from higher animals (vertebrates), contain several introns especially in the TK-domain. The TK-domain of G. cydonium shows similarity with numerous members of receptor as well as nonreceptor TKs. Phylogenetic analysis of the sponge TK-domain indicates that this enzyme branched off first from the common tree of metazoan TK proteins. Consequently, we assume that introns, found in the TK-domains of genes from higher animals, were inserted into these genes after splitting off the sponge taxa from other metazoan organisms (over 600 million years ago). Our results support the view that ancient genes were not ``in pieces.' Received: 8 August 1996 / Accepted: 4 November 1996     

N-Acetylaspartylglutamate Stimulates Metabotropic Glutamate Receptor 3 to Regulate Expression of the GABAAα6 Subunit in Cerebellar Granule Cells

Abstract: We have shown that the vertebrate neuropeptide N-acetylaspartylglutamate (NAAG) meets the criteria for a neurotransmitter, including function as a selective metabotropic glutamate receptor (mGluR) 3 agonist. Short-term treatment of cerebellar granule cells with NAAG (30 µM) results in the transient increase in content of GABA_Aα6 subunit mRNA. Using quantitative PCR, this increase was determined to be up to 170% of control values. Similar effects are seen following treatment with trans-1-aminocyclopentane-1,3-dicarboxylate and glutamate and are blocked by the mGluR antagonists (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine and (2S)-α-ethylglutamic acid. The effect is pertussis toxin-sensitive. The increase in α6 subunit mRNA level can be simulated by activation of other receptors negatively linked to adenylate cyclase activity, such as adenosine A1, α2-adrenergic, muscarinic, and GABA_B receptors. Forskolin stimulation of cyclic AMP (cAMP) levels abolished the effect of NAAG. The change in α6 levels induced by 30 µM NAAG can be inhibited in a dose-dependent manner by simultaneous application of increasing doses of the β-adrenergic receptor agonist isoproterenol. The increase in α6 mRNA content is followed by a fourfold increase in α6 protein level 6 h posttreatment. Under voltage-clamped conditions, NAAG-treated granule cells demonstrate an increase in the furosemide-induced inhibition of GABA-gated currents in a concentration-dependent manner, indicating an increase in functional α6-containing GABA_A receptors. These data support the hypothesis that NAAG, acting through mGluR3, regulates expression of the GABA_Aα6 subunit via a cAMP-mediated pathway and that cAMP-coupled receptors for other neurotransmitters may similarly influence GABA_A receptor subunit composition.     

Anti-Hepatitis A Virus Antibody Response Elicited in Mice by Different Forms of a Synthetic VP1 Peptide

Peptide VP1 (11-25) of the capsid of hepatitis A virus was synthesized by the Fmoc-polyamide solid phase method, and administered to mice in different forms: (1) free, (2) encapsulated in multilamellar liposomes, (3) coupled to keyhole limpet hemocyanin (KHL), and (4) incorporated into a tetrameric branched lysine core. The highest anti-VP1 peptide responses were generated by synthetic peptides entrapped into liposomes and coupled to KLH. No anti-HAV response was generated with the free peptide, while all the other forms induced both anti-HAV and HAV-neutralizing antibodies. Maximum neutralization indices were observed in ascites from mice treated with liposome-entrapped and KLH peptides.     

Plasmid RFLP profiling and DNA homology inThermus isolated from hot springs of different geographical areas

Thirty-four strains belonging to various species of the genus Thermus (T. aquaticus, "T. thermophilus," "T. brockianus," T. scotoductus, and genomic species 2) isolated from hot springs of different geographical areas were examined for plasmid content and restriction fragment length polymorphism (RFLP) of plasmid DNAs. The four strains of the numerical taxonomy cluster E of genomic species 2 did not harbor plasmid DNA. Overall examination of the HindIII-RFLP profiling of plasmid DNA showed considerable variability between and within genomic species, with the exception of presumed clonal isolates. In spite of this heterogeneity, HindIII plasmid digests within a numerical taxonomic cluster gave a subset of restriction fragments of similar or identical length. Strains belonging to genomic species 2 or unclassified isolates from S. Pedro do Sul that harbored plasmid DNA (7 of the 14 strains studied) exhibited strong DNA homology between plasmid regions. No homologous sequences to these plasmid regions were found in chromosomal DNA from strains isolated from S. Pedro do Sul in which no plasmids were detected. The strains belonging to T. scotoductus formed two plasmid DNA homology groups, as estimated by probing with a plasmid fragment that coincided with the two numerical taxonomy clusters proposed previously. Among the other species, homology of plasmid regions was also found between some strains. Strong homology was also found between plasmid regions from some strains of different taxonomic groups, isolated from the same and from different sources, suggesting that these sequences are highly conserved in plasmids present in Thermus. For plasmid-containing strains, results of plasmid RFLP profiling/DNA homology appear promising for the typing of Thermus at the level of biotypes or of individual strains, namely, for monitoring the diversity and frequency of isolates from a particular hot spring. Received: 24 October 1994 / Accepted: 6 March 1995     

Subregional mapping of the human gonadotropin-releasing hormone receptor (GnRH-R) gene to 4q between the markers D4S392 and D4S409

We have isolated nine yeast artificial chromosomes (YACs) containing the gene that encodes the human gonadotropin-releasing hormone receptor (GnRH-R) gene by screening the YAC library of the Centre d'Etude du Polymorphisme Humain (Hôpital Saint-Louis, Paris, France) by the use of the polymerase chain reaction. We defined the location of the GnRH-R gene relative to 4q microsatellite markers D4S392 and D4S409. The genetic positions of these markers on chromosome 4 are 76 and 77 cM, respectively. This location was further established by chromosomal in situ hybridization.     

Evidence from charcoal analysis for environmental change during the interval late Bronze Age to Roman,at the archaeological site of Castro de Penices,N.W. Portugal

Vegetation and environmental change from late Bronze Age to the Roman period in north-west Portugal is reconstructed on the basis of charcoal analyses. The site was occupied by people of the Castrejo culture, i.e. an Iron Age culture that developed in the north-west Iberian peninsula. The pattern of exploitation of natural wood resources by local populations during this period appears to be similar during the three phases of occupation. The frequencies of light-demanding plant species, mostly Leguminosae, testify to considerable destruction and degeneration of the climax woodlands. The preference of particular wood for specific uses, such as roofing, is discussed and the Holocene history of selected trees within the wider region is considered.     

Fluorescence of eosinophil leucocyte granules induced by 1-hydroxy-3,6,8-pyrenetrisulfonate. Visualization of differences in protein isoelectric points

After treatment of horse, rat and human blood smears with alkaline solutions of 1-hydroxy-3,6,8-pyrenetrisulfonate (HPTS), eosinophil leucocyte granules were the unique cell components which showed a bright green fluorescence. When stained with HPTS at pH 10, the whole granule of horse eosinophils showed high emission which strongly diminished after washing or staining in salt solutions or by using blocking methods for amino groups. Using HPTS at pH 12, the fluorescence reaction of house granules was specifically located in the peripheral region, appearing as fluorescent rings. These microscopic observations, which indicate differences in the isoelectric point of proteins within the eosinophil granule, were also confirmed by HPTS staining of protein blots as model substrates. Spectral analysis of HPTS at pH 10 and 12 showed practically identical absorption and emission spectra with peaks at 450 nm and 510 nm, respectively. Our results indicate that mainly ionic binding occurs between cationic proteins and HPTS in alkaline solution, and that the most cationic proteins (with isoelectric points at pH higher than 12) are located in the peripheral annular region of horse eosinophil granules.