Abstract
The obese (ob) gene has been identified through a positional cloning approach; the mutation of this gene causes marked hereditary obesity and diabetes mellitus in mice. We report here the isolation and characterization of the human ob gene. Southern blot analysis demonstrated a single copy of the ob gene in the human genome. The human ob gene spanned approximately 20 kilobases (kb) and contained three exons separated by two introns. The first intron, approximately 10.6 kb in size, occurred in the 5'-untranslated region, 29 base pair (bp) upstream of the ATG start codon. The second intron of 2.3 kb in size was located at glutamine +49. By rapid amplification of 5'-cDNA ends, the transcription initiation sites were mapped 54-57 bp upstream of the ATG start codon. The 172-bp 5'-flanking region of the human ob gene contained a TATA box-like sequence and several cis-acting regulatory elements (three copies of GC boxes, an AP-2-binding site, and a CCAAT/enhancer-binding protein-binding site). By the fluorescence in situ hybridization technique, the ob gene was assigned to human chromosome 7q31.3. This study should establish the genetic basis for ob gene research in humans, thereby leading to the better understanding of the molecular mechanisms underlying the ob gene.
Highlights
The obese gene, an autosomal recessive mutation on mouse chromosome 6, arose spontaneously in the mouse colony at the Jackson Laboratory [1]
Expression of the ob gene is markedly augmented in adipose tissue in several rodent models of genetic obesity (C57BL/6J ob/ob mice [2] and Zucker fatty [4, 6] and Wistar fatty rats [8]) and in rodent models of acquired obesity obtained by pure overfeeding of normal rats1 or by ventromedial lesion to rat hypothalamus [7]
Isolation and Characterization of the Human ob Genomic Fragments—To isolate the human ob gene, ϳ6 ϫ 105 recombinants from a human genomic DNA library in EMBL3 were screened with the 32P-labeled human ob cDNA probe [3]
Summary
Structure among species, and expression of the ob gene is abundant in and specific to adipose tissue in mice [2]. We and others have isolated rat and human ob cDNAs [3,4,5,6,7] and demonstrated that the ob gene is expressed in adipose tissue in a region-specific fashion in rats and humans [3, 4, 8]. Ob gene expression is increased in human obesity in proportion to disease severity [5]. These observations suggest the pathophysiologic roles of the ob gene in the development of obesity. Using the fluorescence in situ hybridization technique, we determined the chromosomal assignment of the human ob gene
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