The GNAS1 gene encodes the ␣ subunit of the G protein G s , which couples receptor binding by several hormones to activation of adenylate cyclase. Null mutations of GNAS1 cause pseudohypoparathyroidism (PHP) type Ia, in which hormone resistance occurs in association with a characteristic osteodystrophy. The observation that PHP Ia almost always is inherited maternally has led to the suggestion that GNAS1 may be an imprinted gene. Here, we show that, although G s ␣ expression (directed by the promoter upstream of exon 1) is biallelic, GNAS1 is indeed imprinted in a promoter-specific fashion. We used parthenogenetic lymphocyte DNA to screen by restriction landmark genomic scanning for loci showing differential methylation between paternal and maternal alleles. This screen identified a region that was found to be methylated exclusively on a maternal allele and was located Ϸ35 kb upstream of GNAS1 exon 1. This region contains three novel exons that are spliced into alternative GNAS1 mRNA species, including one exon that encodes the human homologue of the large G protein XL␣s. Transcription of these novel mRNAs is exclusively from the paternal allele in all tissues examined. The differential imprinting of separate protein products of GNAS1 therefore may contribute to the anomalous inheritance of PHP Ia.

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We describe a screen for new imprinted human genes, and the identification in this way of ZAC (zinc finger protein which regulates apoptosis and cell cycle arrest)/ PLAGL1 (pleomorphicadenoma of the salivary gland gene like 1) as a strong candidate gene for transient neonatal diabetes mellitus (TNDM). To screen for imprinted genes, we compared parthenogenetic DNA from the chimeric patient FD and androgenetic DNA from hydatidiform mole, using restriction landmark genome scanning for methylation. This resulted in identification of two novel imprinted loci, one of which (NV149) we mapped to the TNDM region of 6q24. From analysis of the corresponding genomic region, it was determined that NV149 lies approximately 60 kb upstream of the ZAC / PLAGL1 gene. RT-PCR analysis was used to confirm that this ZAC / PLAGL1 is expressed only from the paternal allele in a variety of tissues. TNDM is known to result from upregulation of a paternally expressed gene on chromosome 6q24. The paternal expression, map position and known biological properties of ZAC / PLAGL1 make it highly likely that it is the TNDM gene. In particular, ZAC / PLAGL1 is a transcriptional regulator of the type 1 receptor for pituitary adenylate cyclase-activating polypeptide, which is the most potent known insulin secretagog and an important mediator of autocrine control of insulin secretion in the pancreatic islet.

We have investigated the thermodynamics and specificity of DNA triplex formation with isothermal titration calorimetry (ITC). The triplex formation between a 23-mer double-stranded homopurine-homopyrimidine and a 15-mer single-stranded homopyrimidine oligonucleotide forming T‚AT and C + ‚GC triads at pH 4.8 is driven by a large negative calorimetric enthalpy change, ∆H cal , of the order of -80 kcal/mol. ∆H cal is strongly temperature dependent, yielding a heat capacity change, ∆C p , of about -1 (kcal/mol)K -1 . The equilibrium association constant, K, obtained from the titration curve is about 9 × 10 7 M -1 at 25°C (binding free energy change, ∆G, is about -11 kcal/mol). Thus, the triplex formation is accompanied by a negative entropy change (∆S ) -245 (cal/mol)K -1 at 25°C). We found that K is insensitive to temperature near room temperature, leading to an apparently small van't Hoff enthalpy change (∆H vH ), in sharp contrast with the large negative ∆H cal . Together, the analyses of the observed temperature dependences of K and ∆H and the large negative ∆C p suggest that the triplex formation is a coupled process between conformational transitions in single-stranded DNA and its binding with double-stranded DNA. The examination of single mismatches in the triplex formation has shown that K and ∆G are not strongly affected by the particular combination of triad sequences (differences in ∆G are within 1.2 kcal/mol). In contrast, single mismatches affected ∆H cal to a greater extent (up to 7-kcal/mol differences). We discuss possible means to enhance specificity in triplex formation, implied by the present findings.