Abstract
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.
Highlights
In screening the regenerating islet-derived cDNA library, we came across a novel gene, reg (Le. regenerating gene), which was expressed in the regenerating islets but not in the normal islets [2,3,4]
The 166-amino acid sequence deduced from the human reg cDNA contained the 144-amino acid sequence of pancreatic stone protein (PSP)’ [6, 7] and the partially determined 45-amino acid sequence of pancreatic thread protein (PTP) [8], suggesting that PSP and PTP are encoded by the reg mRNA
We have identified several molecular forms of reg protein in the human pancreas by immunoblot analysis
Summary
Material.-A genomic DNA library derived from human leukocytes was kindly supplied by Dr Y. E, nucleotide sequence of human reg gene. Isolation and Nucleotide Sequencing of Human reg Gene-The human genomic library, which had been constructed with partial. N&ease Mapping-A go-base synthetic oligodeoxyribonucleotide complementary to the sequence of -19 to 28 plus 330 to 372 of the human reg gene (see Fig. 24) was labeled at the 5’ end with [Y-~‘P]. %P-labeled PstI-XbaI fragment of intron 4 of the human reg gene (see Fig. lA ) as probe. 18 h at 42 ‘C in 50% (v/v) formamide, 5 x SSPE, 5 x Denhardt’s solution, 200 pg/ml salmon testis DNA, and 0.2% SDS using the mixture of 3zP-labeled EcoRI fragments of the human reg cDNA as probe. 1:500 diluted mouse ascites containing the anti-recombinant human reg protein monoclonal antibody in TBS containing 0.5% Tween 20 and 1% nonfat milk at 4 OC overnight. Protein concentrations were determined as described [24]
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