Identification and characterization of a novel transcript of the murine growth hormone receptor gene exhibiting development- and tissue-specific expression.

The growth hormone (GH) receptor gene is characterized by heterogeneity in the 5'-untranslated region (UTR). The technique of 5'-rapid amplification of cDNA ends (RACE) was employed to identify potentially novel 5'-UTRs for the GH receptor gene. One of the RACE clones displayed sequence homology to the human V5-UTR; hence this transcript was designated as L5. Sequence analysis of genomic DNA established that L5 was immediately upstream of exon 2. Northern blot analysis indicated that two bands of sizes congruent with4.8 kb, corresponding to GH receptor mRNA, and congruent with1.5 kb corresponding to GH binding protein mRNA, were detectable in liver, skeletal muscle, kidney and heart but not in brain, spleen, lung or testis. Fluorescent 5'-nuclease real-time RT-PCR based analysis indicated that in the placenta and fetal liver, the L5 transcript represented 10-15% of the GH receptor transcripts. In the adult liver, heart and kidney, the L5 transcript is less abundant accounting for 1-5% of the total GH receptor transcripts. Primer extension and ribonuclease protection assays were performed to identify the major transcription start site at 778 bp from the ATG codon. Transient transfection experiments revealed that the 5'-flanking sequence had promoter activity in rat placental trophoblast (HRP.1), Chinese hamster ovary (CHO) and mouse liver (BNL CL.2) cells. Analysis of expression of the L5 transcript in the non-obese diabetic (NOD) mouse, a model of spontaneous autoimmune diabetes, indicated that the expression of the L5 transcript was decreased in liver and kidney by 80-90 and 40-50%, respectively, but expression remained unchanged in the heart.

Isolation of the regulatory regions and genomic organization of the porcine alpha1,3-galactosyltransferase gene.

BACKGROUND: Alpha1,3-galactosyltransferase (alpha1,3GT) is an enzyme that produces carbohydrate chains termed alphaGal epitopes found in most mammals, although some species of higher primates, including human, are notable exceptions. The evolutionary origin of the lost alpha1,3GT enzyme activity is not yet known, although it has been suggested that the promoter activity of this gene in the ancestors of higher primates was inactivated. METHODS: We used 5'-or 3'-RACE, GenomeWalking, reverse transcriptase polymerase chain reaction (RT-PCR) and dual Luciferase reporter assay for identification of the full-length cDNA, which includes the transcription initiation site and the promoter region of porcine alpha1,3GT gene. RESULTS: The region around exon 1 is guanine and cytosine (GC)-rich (about 70%), comprising a CpG island spanning more than 1.5 kbp. The 5'-flanking region of exon 1 contains multiple transcription factor consensus motifs, including GC-box, SP1, AP2, and GATA-box sites, in the absence of TATA or CAAT-box sequences. The entire gene consists of three 5' noncoding and six coding region exons spanning more than 52 kbp. Detailed analysis of alpha1,3GT transcripts revealed two major alternative splicing patterns in the 5'-untranslated region (5'-UTR) and evidence for minor splicing activity that occurs in a tissue-specific manner. Interspecies comparison of 5'-UTR shows minimal homology between porcine and murine sequences except for exon 2, which suggests that the regulatory regions differ among species. CONCLUSIONS: These observations have important implications for experiments involving genetic manipulation of the alpha1,3GT gene in transgenic animals in terms of promoter utilization, and particularly in genetically engineering cells for the animal cloning technology by nuclear transfer.

Genetics of Type 1 diabetes mellitus.

Type 1 diabetes or insulin-dependent diabetes mellitus (IDDM) is the archetypal example of a T cell-mediated autoimmune disease characterised by selective destruction of a single cell type: the insulin-producing beta-cells of the pancreatic islets of Langerhans. The pathogenic equation for IDDM presents a complex interrelation of genetic and environmental factors, most of which have yet to be identified. Based on the observed familial aggregation of IDDM, it is certain that there is a decided heritable genetic susceptibility for developing autoimmune diabetes. The well-known association of IDDM with certain human histocompatibility leukocyte antigen (HLA) alleles of the major histocompatibility complex (MHC) was a major step toward understanding the role of inheritance in IDDM. Landmark molecular biological investigations of diabetes HLA susceptibility genes provided great potential for insights into the molecular basis for the autoimmune nature of the disease, beginning a story that continues to unfold. Although the association of certain HLA alleles with IDDM is very strong, this genetic locus is estimated to account for less than 50% of genetic contributions to disease susceptibility. The search for non-HLA susceptibility genes has received great attention in recent years. Albeit genome wide searches are wrought with controversy, such studies have suggested the association of numerous non-MHC loci with Type 1 diabetes that will require careful follow-up investigation. Cell biological and genetic functional analyses will provide clues that are indispensable for further progress. The necessary studies include research on immunological abnormalities that are present many years before the clinical onset of Type 1 diabetes.

Sequence analysis of the diabetes-protective human leukocyte antigen-DQB1*0602 allele in unaffected, islet cell antibody-positive first degree relatives and in rare patients with type 1 diabetes.

The human leukocyte antigen (HLA)-DQA1*0102/DQB1*0602/DRB1*1501 (DR2) haplotype confers strong protection from type 1 diabetes. Growing evidence suggests that such protection may be mostly encoded by the DQB1*0602 allele, and we reported that even first degree relatives with islet cell antibodies (ICA) have an extremely low diabetes risk if they carry DQB1*0602. Recently, novel variants of the DQB1*0602 and *0603 alleles were reported in four patients with type 1 diabetes originally typed as DQB1*0602 with conventional techniques. One inference from this observation is that DQB1*0602 may confer absolute protection and may never occur in type 1 diabetes. By this hypothesis, all patients typed as DQB1*0602 positive with conventional techniques should carry one of the above diabetes-permissive variants instead of the protective DQB1*0602. Such variants could also occur in ICA/DQB1*0602-positive relatives, with the implication that their diabetes risk could be significantly higher than previously estimated. We therefore sequenced the DQB1*0602 and DQA1*0102 alleles in all ICA/DQB1*0602-positive relatives (n = 8) previously described and in six rare patients with type 1 diabetes and DQB1*0602. We found that all relatives and patients carry the known DQB1*0602 and DQA1*0102 sequences, and none of them has the mtDNA A3243G mutation associated with late-onset diabetes in ICA-positive individuals. These findings suggest that diabetes-permissive DQB1*0602/3 variants may be very rare. Thus, although the protective effect associated with DQB1*0602 is extremely powerful, it is not absolute. Nonetheless, the development of diabetes in individuals with DQB1*0602 remains extremely unlikely, even in the presence of ICA, as confirmed by our further evaluation of ICA/DQB1*0602-positive relatives, none of whom has yet developed diabetes.