CbbR, a LysR-type transcriptional regulator from Hydrogenophilus thermoluteolus, binds two cbb promoter regions.

The cbbR encoding the LysR-type transcriptional regulator is located downstream of cbbLSQOYA and this gene is located upstream of cbbFPT in divergent transcription. The two promoter regions with LysR-binding sites are located in the cbbL upstream region and in the cbbR-cbbF intergenic region. Electrophoretic mobility shift assays using a cell extract of Escherichia coli harboring a plasmid containing cbbR and the DNA fragments of promoter regions indicated that CbbR binds in both regions. NADPH caused differences in the complex of CbbR and DNA.

Structure of ribulose 1,5-bisphosphate carboxylase/oxygenase gene cluster from a thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus, and phylogeny of the fructose 1,6-bisphosphate aldolase encoded by cbbA in the cluster.

Four genes, cbbO, cbbY, cbbA, and the pyruvate kinase gene (pyk), were found downstream of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes, cbbLS, from a thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus (formerly Pseudomonas hydrogenothermophila). cbbO was similar to norD in the denitrification gene cluster, and cbbY was similar to cbbY from other autotrophic bacteria. cbbA encoded fructose 1,6-bisphosphate aldolase (FBP aldolase); however, CbbA was little similar to other CbbA proteins. When CbbA was overexpressed in Escherichia coli, overproduction of CbbA was detected by SDS-PAGE. However, the cell extract had slightly higher activity than a cell extract of E. coli without cbbA. Phylogenetic analysis showed class II FBP aldolase divided into classes IIA and IIB, and that CbbA from H. thermoluteolus was in class IIA. Activities of RubisCO and FBP aldolase were examined under autotrophic, mixotrophic, and heterotrophic conditions. The activities of the two enzymes were regulated independently.

Isolation and characterization of human reg protein produced in Saccharomyces cerevisiae.

reg was originally identified as a gene expressed during the regeneration of insulin-producing pancreatic beta-cells of the rat. We built an expression vector containing human reg cDNA to drive Saccharomyces cerevisiae to synthesize the reg protein, and purified it from the culture medium. The 144-amino acid sequence of the recombinant protein was consistent with that deduced from the cDNA and genomic DNA sequence except that the signal sequence of 22 amino acids was eliminated, and the amino-terminal residue of the protein was pyroglutamic acid. The secondary structure of the reg protein was predicted by determination of the intramolecular cystine linkage and of alpha-helix and beta-sheet contents.

Nucleotide sequence determination of chicken glucagon precursor cDNA. Chicken preproglucagon does not contain glucagon-like peptide II.

cDNA clones coding for glucagon were isolated from a chicken pancreas cDNA library, and the nucleotide and amino acid sequences were determined. The amino acid sequence of chicken glucagon was HSQGTFTSDYSKYLDSRRAQDFVQWLMST, which was contained in the 151-amino acid long precursor, being preceded by a signal sequence and an amino-terminal peptide (NH2-peptide) and followed by an intervening peptide and a glucagon-like peptide I (GLP-I). Chicken preproglucagon, however, lacked GLP-II and intervening peptide II which have been shown to be contained in mammalian glucagon precursors.

Complete nucleotide sequence of human reg gene and its expression in normal and tumoral tissues. The reg protein, pancreatic stone protein, and pancreatic thread protein are one and the same product of the gene.

We previously identified a novel gene, reg (i.e. regenerating gene), in the screening of a rat regenerating islet-derived cDNA library, and isolated its human cDNA homologue which encodes a 166-amino acid protein (Terazono, K., Yamamoto, H., Takasawa, S., Shiga, K., Yonemura, Y., Tochino, Y., and Okamoto, H. (1988) J. Biol. Chem. 263, 2111-2114). In the present study, we have isolated the human reg gene, determined its complete nucleotide sequence, and examined its expression in human tissues. The functional human reg gene is a single copy gene, spans approximately 3.0 kilobase pairs, and is composed of six exons and five introns. TATA box and CCAAT box-like sequences are located at 27 and 100 base pairs upstream from the transcriptional initiation site. The human reg mRNA was detected predominantly in the pancreas, and at lower levels in the gastric mucosa and the kidney. Furthermore, the reg gene was found to be expressed ectopically in colon and rectal tumors. Immunoblot analysis demonstrated several molecular forms (15-18 kDa) of the reg protein in the pancreas. The 166-amino acid sequence encoded by the human reg gene contains the 144-amino acid sequence of pancreatic stone protein determined by De Caro et al. (De Caro, A. M., Adrich, Z., Fournet, B., Capon, C., Bonicel, J. J., De Caro, J. D., and Rovery, M. (1989) Biochim. Biophys. Acta 994, 281-284) and the partially determined 45-amino acid sequence of pancreatic thread protein (Gross, J., Carlson, R. I., Brauer, A. W., Margolies, M. N., Warshaw, A. L., and Wands, J. R. (1985) J. Clin. Invest. 76, 2115-2126), indicating that the reg protein, pancreatic stone protein, and pancreatic thread protein are simply different names for a single protein deriving from the reg gene.

Expression of reg protein in rat regenerating islets and its co-localization with insulin in the beta cell secretory granules.

Regenerating islets can be induced by the administration of poly(ADP-ribose) synthetase inhibitors to 90% depancreatized rats. In screening a regenerating islet-derived cDNA library, we previously isolated a novel gene. reg (regenerating gene), which encodes a 165-amino acid protein with a 21-amino acid signal sequence. In the present study, we have examined the expression and localization of reg protein in the regenerating islets by immunocytochemical techniques using a monoclonal antibody against a recombinant rat reg protein of 144 amino acids without the signal sequence. Light microscopy examination showed strong immunoreactivity for reg protein in the regenerating islets of the rats at two weeks and two months after 90% pancreatectomy, whereas reg protein was almost undetectable in normal rat islets or in the islets of the rats one year after the pancreatectomy. Almost all the reg protein-positive cells were stained for insulin. By applying the immunogold technique at the ultrastructural level, it was demonstrated that both reg protein and insulin occur in the central granular core of the regenerating Beta cell secretory granules. These results suggest that reg protein is synthesized in and secreted from the regenerating Beta cells and that its expression is closely associated with Beta-cell regeneration.

Expression of the insulinoma gene rig during liver regeneration and in primary cultured hepatocytes.

We have isolated a novel gene, rig (rat insulinoma gene) from rat insulinomas. In the present study, rig was found to be expressed in rat regenerating liver and in primary cultured rat hepatocytes. The level of rig mRNA was increased at the proliferative phase of liver regeneration. In synchronously cultured hepatocytes, the rig mRNA level was elevated at the G1 phase of the cell cycle and the rig-protein was accumulated in the nuclei during the S phase. These results indicated that rig could be involved in a more general way in growth or cell replication.

A novel gene activated in regenerating islets.

Administration of poly(ADP-ribose) synthetase inhibitors such as nicotinamide to 90% depancreatized rats induces regeneration of pancreatic islets, thereby ameliorating the surgical diabetes (Yonemura, Y., Takashima, T., Miwa, K., Miyazaki, I., Yamamoto, H., and Okamoto, H. (1984) Diabetes 33, 401-404). In screening the regenerating islet-derived cDNA library, we came across a novel gene encoding a 165-amino acid protein. The gene was expressed in regenerating islets but not in normal pancreatic islets, insulinomas, or regenerating liver. In 90% depancreatized and nicotinamide-injected rats, the expression of the gene was increased 1 month after the partial pancreatectomy and reached a peak 3 months after the operation. The increase in expression of the gene was temporally correlated with the increase in size of regenerating islets and the decrease in urinary glucose level. The gene was also found to be activated in hyperplastic islets of aurothioglucose-treated mice. Thus, the expression of the gene in both regenerating and hyperplastic islets suggests possible roles for this gene in replication, growth, and maturation of islet beta-cells. We also found that a human pancreas-derived cDNA library contained a homologue to the gene.

Novel gene activated in rat insulinomas.

Insulinomas can be induced in experimental animals by the combined administration of diabetogenic agents with polyadenosine diphosphate (polyADP)-ribose synthetase inhibitors. A complementary DNA (cDNA) library that was constructed from streptozocin-nicotinamide-induced rat insulinomas has been found to contain a novel gene encoding a basic protein of 145 amino acids. The gene was expressed in alloxan-nicotinamide-induced insulinomas as well as in streptozocin-nicotinamide-induced insulinomas but not in normal pancreatic islets or in regenerating islets. This indicates that the activation of the gene designated rig, i.e., rat insulinoma gene, may be a general feature of pancreatic beta-cell transformation.