Structure of the rat glucose-dependent insulinotropic polypeptide receptor gene.

The Glucose-dependent insulinotropic polypeptide receptor (GIPR) is a member of the secretin-vasoactive intestinal polypeptide family of G-protein coupled receptors possessing seven transmembrane domains. We report here the cloning and the exon-intron structure of the rat GIPR gene, along with the identification and characterization of its 5'-flanking region. The coding region of the GIPR gene spans approximately 10.2 kilobases and contains 13 exons. Three additional exons, two encoding either 5' or 3' untranslated sequences and one contained in a novel alternatively spliced mRNA, were identified. The 5'-flanking sequences contained a number of transcription factor binding motifs, including a cAMP response element, an octamer binding site, three SP1 sites and an initiator element. However, neither a CAAT motif nor TATA box were found. Transient transfection assays demonstrated that the 5'-flanking region of the GIPR gene can efficiently promote transcription in RIN38 cells and that deletion of 50 base pairs containing a potential SPI binding sites leads to a 2.4-fold loss of transcriptional activity. In addition, transient transfection experiments comparing the relative promoter activities of 5'-flanking sequences of the GIPR gene in RIN38 and rat-2 cells suggests that distal negative regulatory sequences may control cell-specific expression.

Cell-specific expression of the glucose-dependent insulinotropic polypeptide gene in a mouse neuroendocrine tumor cell line.

Glucose-dependent insulinotropic polypeptide (GIP) is a 42-amino acid gastrointestinal regulatory peptide that, in the presence of glucose, stimulates insulin secretion. GIP is expressed in K cells of the small intestine and in cells of the submandibular salivary gland. Using a rat GIP cDNA as a specific probe, we screened a number of established cell lines for the expression of GIP mRNA. STC-1 cells, a cell line derived from a mouse neuroendocrine tumor, were found to express high levels of GIP mRNA. GIP-specific transcripts were not detected in other cell lines tested, which included cells of intestinal, salivary, and endocrine origin. Analysis of GIP-luciferase fusions identified two promoters, a distal and a proximal promoter, upstream of the translation initiation codon for GIP. The distal promoter, located upstream of position +1, corresponds to the principal promoter of the GIP gene and can promote cell-specific transcription. Sequential deletion and site-directed mutational analysis of the distal promoter demonstrated that the sequence between -193 and -182 determines cell-specific expression of GIP. Contained in this region is a consensus GATA motif, suggesting that a member of the GATA family of DNA-binding proteins is involved in the cell-specific regulation of the GIP gene.

A human gene homologous to the formin gene residing at the murine limb deformity locus: chromosomal location and RFLPs.

The murine limb deformity (ld) locus resides on mouse chromosome 2 and gives rise to a recessively inherited, characteristic limb deformity/renal aplasia phenotype. In this locus in the mouse, a gene, termed the "formin" gene, has been identified which encodes an array of differentially processed transcripts in both adult and embryonic tissues. A set of these transcripts are disrupted in independent mutant mouse ld alleles. We wish to report the isolation of a human genomic clone which is homologous to the mouse formin gene by virtue of sequence comparison and expression of conserved exons. Among human fetal tissues analyzed, the kidney appears to be a major site of expression. This human gene, LD, maps to chromosome 15q11----qter in mouse human somatic cell hybrids and, specifically, to 15q13----q14 by chromosomal in situ hybridization. This localization establishes both LD and beta 2-microglobulin as syntenic genes on mouse chromosome 2 and human chromosome 15 and implies the interspecies conservation of the region between them. In addition, we identify in the human locus two frequently occurring DNA polymorphisms which can be used to test the linkage of LD to known human dysmorphoses.