Biomedical subjects
Publications and source records attributed to K G Xanthopoulos.
The mouse gene Cebp, encoding the DNA-binding protein C/EBP, has been localized to the proximal region of chromosome 7 by determining the strain distribution patterns of a restriction fragment length polymorphism among the BXD and AKXL recombinant inbred mouse lines.
Pupae of the moth Hyalophora cecropia respond to an injection of live bacteria by the production of a potent antibacterial activity. The broad-spectrum property of this activity is due chiefly to two small proteins, cecropins A and B. Sequences of the proteins showed them to be homologous and to contain 37 and 35 amino acid residues respectively. The subsequent isolation of two cDNA clones for cecropin B showed that this protein is made as a prepro molecule composed of 62 amino acid residues. We have now prepared a genomic bank and studied four genomic clones for cecropin B. The coding regions were found in two neighbouring BglII fragments, one 0.79 kb and another varying in size from 3.1 kb to 4.9 kb for different clones. One transcriptional unit for preprocecropin B was sequenced and found to be 1035 bp long with a single intron, 514 bp in size. A conserved, insect specific cap site, ATCATTC, was identified by S1 mapping and primer extension experiments. Indications were found for the presence of multigene families and multicopy genes.
Four different regulatory sites required for transcriptional stimulation by the enhancers of two unrelated liver-specific genes alpha 1-antitrypsin and transthyretin appear to bind the same nuclear protein that is found mainly in the liver. Such proteins may provide a basis for a coordinated, hepatocyte-specific control of gene transcription.
We have made use of RNA.RNA in situ hybridization to study the presence of cells producing mRNA for interleukin 4 (IL-4) in the developing thymus, spleen, and T-cell line 2.19. Approximately 1 of 300-400 spleen cells expressed detectable IL-4 mRNA 24 hr after their stimulation by the lectin concanavalin A. Spleen cells were also induced to express mRNA for IL-4 by stimulation with alloantigens. Splenocytes producing mRNA for IL-4 were detected 4 hr after stimulation by concanavalin A; the response peaked at approximately equal to 24 hr and was undetectable by 72 hr. Cyclosporin A inhibited the synthesis of IL-4 mRNA in the T-cell line 2.19, which had been induced by concanavalin A. Approximately 1 of 10 fetal thymocytes at day 14 of gestation expressed mRNA for IL-4 after their stimulation by phorbol 12-myristate 13-acetate and ionomycin. Both the frequency of fetal thymocytes expressing IL-4 mRNA and the amount of mRNA for IL-4 synthesized per cell sharply decreased at day 16 of gestation, and less than 1 of 1800 fetal thymocytes at day 18 of gestation expressed detectable IL-4 mRNA. Our results define the relative frequency of cells capable of expressing IL-4 mRNA after stimulation in vitro in the spleen and in the developing thymus. The data strongly argue for an important role of IL-4 in growth and differentiation of lymphoid cells, notably during T-cell development within the thymus.
Double-stranded oligodeoxynucleotides that represent protein binding sites in the regulatory regions of the mouse genes encoding transthyretin (TTR) and alpha 1-antitrypsin (alpha 1-AT) bound a nuclear protein factor(s) found mainly in hepatocytes. A site in the regulatory region of the gene encoding rat serum albumin and, surprisingly, a region in the simian virus 40 enhancer also bind the same factor. Oligodeoxynucleotide affinity chromatography (with one of the TTR binding sites) allowed a 500-fold purification of the protein. The purified protein protected similar portions of all the regulatory regions, as well as the simian virus 40 core C enhancer element, from digestion with DNase I. A DNA-binding protein previously purified from liver by virtue of its ability to bind to several virus enhancer sequences also binds to TTR, alpha 1-AT, and albumin regulatory sites. Thus, all these binding sites, which contain only minimal sequence similarity, may bind to a single protein, or a similar family of proteins, that activates liver-specific transcription of coordinately expressed genes.
We have previously described the isolation and characterization of genomic clones corresponding to the mouse alpha 1-antitrypsin gene (Krauter et al., DNA 5:29-36, 1986). In this report, we have analyzed the DNA sequences upstream of the RNA start site that direct hepatoma cell-specific expression of this gene when incorporated into recombinant plasmids. The 160 nucleotides 5' to the cap site direct low-level expression in hepatoma cells, and sequences between -520 and -160 bp upstream of the RNA start site functioned as a cell-specific enhancer of expression both with the alpha 1-antitrypsin promoter and when combined with a functional beta-globin promoter. Within the enhancer region, three binding sites for proteins present in hepatoma nuclear extracts were identified. The location of each site was positioned, using both methylation protection and methylation interference experiments. Each protein-binding site correlated with a functionally important region necessary for full enhancer activity. These experiments demonstrated a complex arrangement of regulatory elements comprising the alpha 1-antitrypsin enhancer. Significant qualitative differences exist between the findings presented here and the cis-acting elements operative in regulating expression of the human alpha 1-antitrypsin gene (Ciliberto et al., Cell 41:531-540, 1985; De Simone et al., EMBO J. 6:2759-2766, 1987).
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The amino acid and cDNA sequences of lysozyme from the giant silk moth Hyalophora cecropia have been determined. This enzyme is one of several immune proteins produced by the diapausing pupae after injection of bacteria. Cecropia lysozyme is composed of 120 amino acids, has a mol. wt. of 13.8 kd and shows great similarity with vertebrate lysozymes of the chicken type. The amino acid residues responsible for the catalytic activity and for the binding of substrate are essentially conserved. Three allelic variants of the Cecropia enzyme are identified. A comparison of the chicken and the Cecropia lysozymes shows that there is a 40% identity at both the amino acid and the nucleotide level. Some evolutionary aspects of the sequence data are discussed.
Diapausing pupae of Cecropia respond to a bacterial infection by the selective synthesis of RNA and 15-20 hemolymph proteins. Of these we have purified lysozyme and two classes of antibacterial proteins called cecropins and attacins. The primary structure has been determined for the lysozyme, one attacin and five cecropins. We have also prepared a cDNA bank, isolated and sequenced clones corresponding to the lysozyme, the two main attacins and one cecropin. The results of these structural studies are briefly summarized. Finally we review the solid phase synthesis of cecropin A and B and 9 analogs of cecropin A.
Two cDNA clones containing coding information for cecropin B from the Cecropia moth (Hyalophora cecropia) were identified by means of a synthetic probe. Sequencing of the two inserts showed that cecropin B is processed from a 62-amino acid residue precursor molecule including a 26-residue leader peptide and a COOH-terminal glycine residue. The latter presumably donates the nitrogen of the amide group present on the COOH-terminal leucine residue of the mature cecropin B. The sequence deduced for the mature cecropin B differed in the COOH-terminal region from the tentative structure previously determined by carboxypeptidase digestion. To settle the discrepancy, cecropin B was synthesized according to the cDNA sequence with an amidated COOH-terminal leucine. Natural and synthetic cecropin B were found to be indistinguishable with respect to electrophoretic mobility and antibacterial activity against seven different bacteria. The COOH-terminal tetrapeptides were isolated from both natural and synthetic cecropin B and found to be indistinguishable. The correct sequence for cecropin B is (formula; see text).
The Cecropia moth has three known classes of antibacterial immune proteins, attacins, lysozyme and cecropins (earlier referred to as P5, P7 and P9, respectively). Six attacins with different isoelectric points have been purified. The N-terminal sequences for five of these forms imply that only two different genes exist. We have now isolated and sequenced two cDNA clones, one for the basic attacin and one for the acidic form. The two mature proteins show 76% homology at the nucleotide level, while the regions beyond the stop codons are 36% homologous. The differences in the content of aspartic acid accounts for the difference in net charge between the acidic and basic attacin. Further differences in charge can be obtained by post-translational removal of a lysine-containing tetrapeptide at the C-terminal end of the two proteins. Evidence for a prepro form of the basic attacin is presented.