Characterization of C14orf4, a novel intronless human gene containing a polyglutamine repeat, mapped to the ARVD1 critical region

Within the ARVD1 (arrhythmogenic right ventricular dysplasia/cardiomyopathy, type 1) critical region, mapped to 14q24.3, we detected an intronless gene of 4859 bp, predominantly expressed in the heart tissue. This gene encodes a 796-amino-acid, proline-rich protein showing polyglutamine and polyalanine tracks with variable length at the N-terminus and a C3HC4 RING finger domain at the C-terminus. CREB and AP-2 binding sites are present in the promoter region. The 5' flanking region contains neither a TATA box nor a CAAT box, but it is high in GC content and includes several Sp1 binding sites. Protein similarity searches revealed a significant match between the C-terminus and a human hypothetical protein, whose gene is located on the chromosome 19 long arm. The predicted protein shows PEST sequences, suggesting its rapid degradation. The novel intronless gene, provisionally named C14orf4 and probably encoding a nuclear protein, was excluded from being the ARVD1 gene.     

Isolation and characterisation of a cDNA encoding the precursor for a novel member of the acyl-CoA dehydrogenase gene family.

A gene encoding the precursor for a novel member of the human acyl-CoA dehydrogenase (ACD) gene family has been isolated which maps to human chromosome 11q25. The cDNA contains an open reading frame of 1248 nucleotides encoding a predicted 415-amino-acid peptide, and shares considerable sequence similarity with other members of the ACD family.     

Evolution of the vertebrate glucose-dependent insulinotropic polypeptide (GIP) gene

The glucose-dependent insulinotropic polypeptide (GIP) gene is believed to have originated from a gene duplication event very early in vertebrate evolution that also produced the proglucagon gene, yet so far GIP has only been described within mammals. Here we report the identification of GIP genes in chicken, frogs, and zebrafish. The chicken and frog genes are organized in a similar fashion to mammalian GIP genes and contain 6 exons and 5 introns in homologous locations. These genes can also potentially be proteolytically processed in identical patterns as observed in the mammalian sequences that would yield a GIP hormone that is only one amino shorter than the mammalian sequences due to the removal of an extra basic residue by carboxypeptidase E. The zebrafish GIP gene and precursor protein is shorter than other vertebrate GIP genes and is missing exon 5. The predicted zebrafish GIP hormone is also shorter, being only 31 amino acids in length. The zebrafish GIP hormone is similar in length to the proglucagon-derived peptide hormones, peptides encoded from the gene most closely related to GIP. We suggest that the structure of zebrafish GIP is more similar to the ancestral gene, and that tetrapod GIP has been extended. The mammalian GIP hormone has also undergone a period of rapid sequence evolution early in mammalian evolution. The discovery of a conserved GIP in diverse vertebrate suggests that it has an essential role in physiology in diverse vertebrates, although it may have only recently evolved a role as an incretin hormone.     

Molecular cloning of rat glucose-dependent insulinotropic peptide (GIP).

A cDNA clone encoding glucose-dependent insulinotropic peptide (GIP) was identified that consisted of 34 bp of 5' untranslated sequence, an open reading frame of 432 bp and 115 bp in the 3' untranslated region. The deduced amino acid sequence revealed a 144 amino acid preprohormone consisting of a 43 amino acid N-terminal extension including a signal peptide, a 42 amino acid hormone, and a 59 amino acid C-terminal extension. Rat GIP differs from the human hormone by two amino acid substitutions: arginine for histidine at position 18 and leucine for isoleucine at position 40. A single mRNA from small intestine of approximately 800 bases was identified on Northern blot analysis in equivalent amounts in proximal and distal small intestine.     

Insulin is secreted upon glucose stimulation by both gastrointestinal enteroendocrine K-cells and L-cells engineered with the preproinsulin gene

Transgenic mice carrying the human insulin gene driven by the K-cell glucose-dependent insulinotropic peptide (GIP) promoter secrete insulin and display normal glucose tolerance tests after their pancreatic p-cells have been destroyed. Establishing the existence of other types of cells that can process and secrete transgenic insulin would help the development of new gene therapy strategies to treat patients with diabetes mellitus. It is noted that in addition to GIP secreting K-cells, the glucagon-like peptide 1 (GLP-1) generating L-cells share/ many similarities to pancreatic p-cells, including the peptidases required for proinsulin processing, hormone storage and a glucose-stimulated hormone secretion mechanism. In the present study, we demonstrate that not only K-cells, but also L-cells engineered with the human preproinsulin gene are able to synthesize, store and, upon glucose stimulation, release mature insulin. When the mouse enteroendocrine STC-1 cell line was transfected with the human preproinsulin gene, driven either by the K-cell specific GIP promoter or by the constitutive cytomegalovirus (CMV) promoter, human insulin co-localizes in vesicles that contain GIP (GIP or CMV promoter) or GLP-1 (CMV promoter). Exposure to glucose of engineered STC-1 cells led to a marked insulin secretion, which was 7-fold greater when the insulin gene was driven by the CMV promoter (expressed both in K-cells and L-cells) than when it was driven by the GIP promoter (expressed only in K-cells). Thus, besides pancreatic p-cells, both gastrointestinal enteroendocrine K-cells and L-cells can be selected as the target cell in a gene therapy strategy to treat patients with type 1 diabetes mellitus.     

Expression of recombinant human glucose-dependent insulinotropic polypeptide in Escherichia coli by sequence-specific proteolysis of a protein A fusion protein

Glucose-dependent insulinotropic polypeptide (GIP) is a forty-two amino acid hormone that stimulates the secretion of insulin from the pancreatic B-cells in the presence of elevated glucose concentrations. The human GIP gene with the human A-fibrinopeptide sequence was synthesized and linked to the Staphylococcus aureus protein A gene in the vector pRIT2T. This plasmid was expressed in Escherichia coli, and the resulting fusion protein consisted of three domains: protein A for ease of purification, fibrinopeptide sequence for thrombin cleavage and human GIP. The GIP was subsequently cleaved from the fusion protein with -thrombin. The identity of the recombinant human GIP was confirmed by SDS-PAGE, ELISA, HPLC and amino-terminal amino acid sequence analysis. This recombinant product was shown to have comparable insulinotropic activity to porcine GIP in the isolated perfused pancreas.     

Typical Danish Caucasian type 2 diabetic patients do not commonly carry genetic variants in GIP and GLP-1 encoding regions of the proGIP and proglucagon genes

BACKGROUND: The enteroinsular-axis is abnormal in type 2 diabetics, which contributes to the diabetic phenotype. The effect of the incretin hormone gastric inhibitory polypeptide (GIP) and the secretion of the incretin hormone glucagon-like peptide-1 (GLP-1) are thus greatly diminished. The explanation for these changes could be changes in the structure of either of the hormones or their receptors. Thus, the aim of this study was to study the occurrence of genetic variants in the GIP and GLP-1 encoding regions of the proGIP and proglucagon genes in type 2 diabetic patients and matched control subjects. METHODS AND RESULTS: Genomic DNA was extracted from buffy coats from 12 Caucasian type 2 diabetics and 12 healthy subjects, matched with respect to sex, age and BMI. The GIP and GLP-1 sequences were amplified using specific primers using the polymerase chain reaction (PCR). The amplified products were then sequenced. No germ-line mutations were identified in the GIP and the GLP-1 encoding regions of the proGIP and proglucagon genes in either the type 2 diabetic or the control subjects. CONCLUSIONS: The perturbed incretin effect in type 2 diabetics is not commonly caused by genetic variants in either the GIP or the GLP-1 encoding genes in type 2 diabetics.     

Genomic organization and chromosomal location of the mouse vasoactive intestinal polypeptide 1 (VPAC1) receptor.

The gene encoding the mouse vasoactive intestinal polypeptide type 1 (VPAC1) receptor was cloned, and its structural organization was determined. The gene (Vipr1) is more than 16 kb in length and is divided into 13 exons. The 5'-flanking region is highly GC-rich and lacks an apparent TATA box, but contains a CCAAT box, three potential Sp1-binding sites, and two potential AP-2-binding sites. Promoter analysis of the 5'-flanking region of Vipr1 using a luciferase gene reporter system revealed that the isolated 5'-flanking region has functional promoter activity. The mouse Vipr1 gene is encoded by a single gene, which was mapped to the distal region of mouse chromosome 9. This region is syntenic with human chromosome 3p, where the human VPAC1 receptor gene has been mapped.     

The vitamin D-responsive element in the human osteocalcin gene. Association with a nuclear proto-oncogene enhancer

A vitamin D-responsive element (VDRE) locus within the 5' region of the human osteocalcin gene promoter contains a steroid response-like half-site immediately proximal to a consensus site for the AP-1 nuclear oncogene family. In the studies described here, internal mutagenesis of the osteocalcin promoter coupled to functional assays reveal that the interaction of the vitamin D receptor is limited to the proximal region of the VDRE locus. Mutations within the distal AP-1 consensus site reduce the basal activity of the promoter but have little effect on vitamin D inducibility. The absolute level of promoter activity induced by hormone, however, is dramatically reduced in the absence of an intact AP-1 site suggesting a functional synergism between the receptor and AP-1-related proteins. In vitro receptor-DNA binding studies confirm the lack of requirement for the distal component in receptor binding. These results suggest that the osteocalcin VDRE is limited to 15 nucleotides closely juxtaposed to a distal functional AP-1 site. The close association of the two sites may lead to proto-oncogene and steroid receptor interactions that result in interesting functional consequences.     

Cloning of the pig aminopeptidase N gene. Identification of possible regulatory elements and the exon distribution in relation to the membrane-spanning region

We have isolated four lambda-phages covering the complete pig aminopeptidase N/CD13 gene. The sequence of 2.85 kbp encompasses 1.18 kbp of the 5' upstream region and 1.67 kbp of the structural gene. In the promoter region we find a TATA box and potential binding sites for CTF-1/NF-1 and AP-2. By sequence comparisons we have found three domains showing similarity to promoter regions of the genes encoding human alpha 1-antitrypsin and human intestinal alkaline phosphatase. The gene sequence includes the first three exons and two introns. It shows that a single exon encodes the cytoplasmic tail, the membrane anchor and the junctional peptide.