Human secretin (SCT): gene structure, chromosome location, and distribution of mRNA.

Secretin is an endocrine hormone that stimulates the secretion of bicarbonate-rich pancreatic fluids. Recently, it has been discussed that secretin deficiency may be implicated in autistic syndrome, suggesting that the hormone could have a neuroendocrine function in addition to its role in digestion. In the present study, the human secretin gene (SCT) was isolated from a bacterial artificial chromosome genomic library. SCT contains four exons, with the protein coding regions spanning 713 bp of genomic DNA. Human SCT is similar structurally to the secretin genes of other species. Amino acid conservation, however, is most pronounced within the exon encoding the biologically active mature peptide. Northern blot analysis shows that human SCT transcripts are located in the spleen, intestinal tract, and brain. Radiation hybrid mapping places the SCT locus on chromosome 11p15.5.

Identification of INSL6, a new member of the insulin family that is expressed in the testis of the human and rat.

A new member of the insulin gene family (INSL6) was identified from an Expressed Sequence Tag database through a search for proteins containing the insulin family B-chain cysteine motif. Human and rat INSL6 encoded polypeptides of 213 and 188 amino acids, respectively. These orthologous sequences contained the B-chain, C-peptide, and A-chain motif found in other members of the insulin family. Human INSL6 was 43% identical to human relaxin H2 in the B- and A-chain regions. As with other family members, human and rat INSL6 had predicted dibasic sequences at the junction of the C-peptide and A-chain. Human INSL6 sequence had an additional dibasic site near the C-terminus of the A-chain. The presence of a single basic residue at the predicted junction of the B-chain and C-peptide suggests that multiple prohormone convertases are required to produce the fully mature hormone. INSL6 was found to be expressed at high levels in the testis as determined by Northern blot analysis and specifically within the seminiferous tubules in spermatocytes and round spermatids as detected by in situ hybridization analysis. Radiation hybrid mapping placed the human INSL6 locus at chromosome 9p24 near the placenta insulin-like homologue INSL4 and the autosomal testis-determining factor (TDFA) locus.

Identification of INSL5, a new member of the insulin superfamily.

A new member of the insulin gene superfamily (INSL5) was identified by searching EST databases for the presence of the conserved insulin B-chain cysteine motif. Human and murine INSL5 are both polypeptides of 135 amino acids, matching the classical signature of the insulin superfamily. Through the B- and A-chain regions, human INSL5 has 48% identity to shark relaxin, 40% identity to human relaxin, and 34% identity to human Leydig insulin-like factor. Northern blot analysis detected expression of human INSL5 in rectal, colon, and uterine tissue and of murine INSL5 only in thymic tissue. Using quantitative RT-PCR, expression of murine INSL5 was detected in the highest quantity in colon followed by thymus, and minimal expression was seen in testis. By radiation hybrid mapping and the use of surrounding markers, human INSL5 maps to chromosome 1 in the 1p31.1 to 1p22.3 region.

Recombinant ob protein reduces feeding and body weight in the ob/ob mouse.

To determine whether the product of the recently cloned ob gene functions as an adipose-related satiety factor, recombinant murine ob protein was administered intraperitoneally to ob/ob mice. Monomeric ob protein given as single morning injections to groups of three animals at seven doses ranging from 5 to 100 micrograms reduced 24-h chow consumption in a dose-dependent manner from values of 81 +/- 6.8% of control (10-micrograms dose, P = 0.04) to 29 +/- 7.7% of control (100-micrograms dose, P < 0.0001). Daily injections of 80 micrograms of ob protein into six ob/ob mice for 2 wk led to an 11 +/- 1.6% decrease in body weight (P = 0.0009) and suppressed feeding to 26 +/- 4.9% of baseline (P < 0.0001), with significant reduction of serum insulin and glucose levels. The effect of recombinant ob protein on feeding was not augmented by cofactors secreted by adipose tissue, nor did exposure of adipose tissue to ob protein affect intracellular ob mRNA levels. Posttranslational modification of ob protein was not required for activity; however, addition of a hexahistidine tag to the amino terminus of the mature ob protein resulted in prolonged suppression of feeding after injection into ob/ob mice. These results demonstrate a direct effect of the ob protein to suppress feeding in the ob/ob mouse and suggest that this molecule plays a critical role in regulating total body fat content.

Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo.

The major regulator of circulating platelet levels is believed to be a cytokine termed thrombopoietin. It is thought to be a lineage-specific cytokine affecting the proliferation and maturation of committed cells resulting in the production of megakaryocytes and platelets. Despite considerable efforts by a number of laboratories, the unequivocal identification of thrombopoietin has proven elusive. Here we report the functional cloning of a murine complementary DNA encoding a ligand for the receptor encoded by the c-mpl proto-oncogene (c-Mpl). The encoded polypeptide has a predicted molecular mass of 35,000 (M(r) 35K). The protein has a novel two-domain structure with an amino-terminal domain homologous with erythropoietin and a carboxy-terminal domain rich in serine, threonine and proline residues and containing seven potential N-linked glycosylation sites. Intraperitoneal injections of mice with recombinant protein increase circulating platelet levels by greater than fourfold after 7 days. These results along with those presented in the accompanying report strongly suggest that the ligand for c-Mpl is thrombopoietin.

Complete cDNA encoding human phospholipid transfer protein from human endothelial cells.

Phospholipid transfer protein, with an apparent molecular mass of 81 kDa, was purified from human plasma. The NH2-terminal amino acid sequence of a 51-kDa proteolytic fragment obtained from phospholipid transfer protein allowed degenerate primers to be designed for polymerase chain reaction and the eventual isolation of a full-length cDNA from a human endothelial cDNA library. The cDNA is 1,750 base pairs in length and contains an open reading frame of 1,518 nucleotides encoding a leader of 17 amino acids and a mature protein of 476 residues. Northern blot analysis shows a single mRNA transcript of approximately 1.8 kilobases with a wide tissue distribution. The gene was mapped to chromosome 20 using a human/rodent somatic cell hybrid mapping panel. Phospholipid transfer protein was found to be homologous to human cholesteryl ester transfer protein, human lipopolysaccharide-binding protein, and human neutrophil bactericidal permeability increasing protein (20, 24, and 26% identity, respectively).