Masashi Kawaichi

The Drosophila homolog of the immunoglobulin recombination signal-binding protein regulates peripheral nervous system development

The J kappa RBP binds to the immunoglobulin recombination signal sequence flanking the kappa-type J segment. We previously isolated the highly conserved homolog of the J kappa RBP gene from D. melanogaster, which is not thought to have immunoglobulin molecules. Using many deficiency mutants and in situ hybridization, we mapped the Drosophila J kappa RBP gene in a region containing two recessive lethal mutations, i.e., br26 and br7, which shows the dominant Suppressor of Hairless (Su(H)) phenotype in heterozygotes. All six Su(H) alleles analyzed at the DNA level contained mutations in the Drosophila J kappa RBP gene. Since the Su(H) mutation affects peripheral nervous system development, the Drosophila J kappa RBP gene product is involved in gene regulation of peripheral nervous system development. The results also imply that the immunoglobulin recombination signal sequence and the target sequence of the Drosophila J kappa RBP protein might have a common evolutionary origin.

ADP-ribosylation reactions.

Publisher Summary ADP-ribosylation involves a group of posttranslational modifications of proteins in which the ADP-ribose moiety of NAD is transferred to various protein acceptors. The ADP-ribose transferred remains monomeric or is polymerized, which allows the classification of mono(ADP-ribosyl)ation and poly(ADP-ribosyl)ation. This chapter discusses the various mono- and poly(ADP-ribosyl)ation reactions. Many of the enzymes catalyzing mono(ADP-ribosyl)ation are microbial toxins such as diphtheria toxin, cholera toxin, and Escherichia coli enterotoxin, whereas their targets are eukaryotic enzymes such as elongation factor 2 and a guanine nucleotide regulatory component of the adenylate cyclase system. On the other hand, poly(ADP-ribosyl)ation involves almost exclusively enzymes as well as acceptors of eukaryotic origin. Mono- and poly(ADP-ribosyl)ation are further different in the reversibility of the reaction and the nature of ADP-ribosyl protein linkages (N-glycosides versus O-glycosides). The chapter discusses the present knowledge of and recent results on poly(ADP-ribosyl)ation reactions. The biosynthesis of poly(ADP-ribosyl) protein is reconstituted in vitro with purified synthetase preparations.

POLY(ADP-RIBOSE) AND ADP-RIBOSYLATION OF PROTEINS

Publisher Summary This chapter analyzes poly(ADP-ribose) and ADP-ribosylation of proteins. Poly(ADP-ribose) and the ADP-ribosylation of proteins constitute a novel type of covalent modification of proteins. They are ubiquitously distributed in nature and are implicated in the regulation of cell proliferation, protein synthesis, and DNA as well as RNA metabolism. This type of post-translational modification is unique because NAD, nicotinamide adenine dinucleotide, whose primary function is an electron carrier in biological oxidation, invariably provides the ADP-ribosyl moiety, which is transferred on to a protein molecule. The ADP-ribosyl unit thus, covalently attached to a protein acceptor is present as either a monomer or a polymer as in the case of the nuclear system. This chapter also summarizes developments in this field of research and some experimental results. It reviews briefly, mono ADP-ribosylation of proteins, in which only a single ADP-ribosyl moiety is transferred to a protein acceptor. It also covers a more complex reaction, poly(ADP-ribose) in nuclei, in which the ADP-ribosyl units are polymerized.

A new method for oligo(ADP-ribose) fractionation according to chain length☆

Abstract A new method was developed to separate mono- and oligo-(ADP-ribose) with chain lengths below 11 ADP-ribose units by size difference of one ADP-ribose residue. The separation was performed on a DEAE-cellulose column by elution with a NaCl gradient (0–0.3 M ) in the presence of 7 M urea at pH 7.6. Using this method, the chain length distribution of oligo(ADP-ribose) molecules attached to histones by incubation of isolated nuclei with radioactive NAD was determined. The average chain length estimated from this distribution coincided exactly with the value obtained by the phosphodiesterase digestion method, suggesting that the oligomers were synthesized directly on histones and not elongated from pre-existing ADP-ribose.

Cloning and Characterization of a Protein Binding to the Jκ Recombination Signal Sequence of Immunoglobulin Genes

A protein with molecular weight of 60,000 that binds to the recombination signal sequence (RS) of the immunoglobulin J kappa segment was purified from the nuclear extract of a murine pre B cell line 38B9. This binding protein was found in lymphoid cell lines but not in non-lymphoid cell lines. The Kd value of the J kappa RS binding protein to the J kappa RS was 1 nM. The cDNA clone (RBP-2) was isolated based on partial amino-acid sequence of this protein. This cDNA encodes 526 amino-acid residues, and its sequence does not show extensive overall homology with any known proteins, but displays an interesting homology to a 40-residue region that is conserved among a subset of site specific recombinase (integrase family).

Germline transcripts of the immunoglobulin heavy-chain and T cell receptor genes in a murine hematopoietic stem cell line LyD9 and its derivative cell lines

We compared germline transcript levels of immunoglobulin heavy chain and T cell receptor (TcR) genes in a murine hematopoietic stem cell line, LyD9, and its derivative cell lines. LyD9 cells can be induced to differentiate into at least three lineages, namely, B lymphocyte, macrophage, and granulocyte lineages. Although C mu transcripts were found in stem cells to B lymphocytes, other myeloid-committed cells also expressed significant amounts of C mu transcripts. Germline TcR transcripts did not show good correlation with differentiation potential and stages of hematopoietic cells. During this search we identified a novel germline transcript containing the JH-C microliter sequence in LyD9 and some of its derivative cells. Expression of mRNAs for immunoglobulin- and TcR-associated molecules (lambda 5, MB1 and CD3 delta) was widespread except for lambda 5 mRNA. Among three mRNAs encoding putative recombinase proteins, RAG-1 and RAG-2 mRNAs were not expressed in any cell lines tested, while RBP-2 mRNA was expressed ubiquitously.

Unexpected Biological Effects of the Deregulated IL-2/IL-2 Receptor System on the Lymphocyte Development

Interleukin-2 (IL-2), a glycoprotein formerly called T-cell growth factor, is secreted by peripheral helper T cells when they recognize antigenic fragments in association with protein products of the major histocompatibility complex (MHC). Secreted IL-2 then binds to its receptor expressed on antigen-stimulated peripheral T cells and induces their proliferation (Smith, 1988). The interleukin-2 receptor (IL-2R) consists of two polypeptide chains, namely the light (L) chain (p55) and the heavy (H) chain (p75) (Tsudo et al., 1986; Robb et al., 1987; Teshigawara et al., 1987). The L and the H chains bind IL-2 with low (Kd 10 nM) and intermediate (Kd 100 pM) affinities, respectively. IL-2 first binds to the L chain and the IL-2-L chain complex then quickly associates with the H chain on the cell surface to form the ternary complex of IL-2, the L chain and the H chain, which gives rise to the high-affinity binding site (Kd 10 pM) and transduces the growth signal (Kondo et al., 1987; Ogura et al., 1988; Saito et al., 1988). The L chain, which alone cannot transduce the growth signal (Sabe et al., 1984; Greene et al., 1985), is expressed transiently on peripheral T cells only when they are stimulated by antigens or mitogens (Waldmann, 1986). On the other hand, the H chain is able to transduce the growth signal by itself at a higher concentration of IL-2 (Siegel et al., 1987; Tsudo et al., 1987; Wang and Smith, 1987) and constitutively expressed on T cells in a limited number (Sharon et al., 1987; Nishi et al., 1988).

Automodification of Poly(ADP-ribose) Synthetase and DNA Repair

Poly (ADP-Ribose) is a unique homopolymer composed of a linear sequence of repeating ADP-ribose units that are derived from NAD, nicotinamide-adenine dinucleotide. The ADP-ribose moiety is linked together by ribose 1”→2’ ribose glycosidic bonds and the terminal ribose is covalently attached to an acceptor protein (Hayaishi and Ueda, 1977).