(Received for publication, July 24, 1995; and in revised form, November 16, 1995)
From the
The human gene encoding the homolog of the mouse obese (ob) gene was isolated and partially characterized. The human ob gene consists of three exons and two introns and spans about 18 kilobase pairs (kb), encoding a 3.5-kb cDNA. A 3-kb 5`-flanking region of the gene was cloned and transient transfection assay with luciferase reporter confirmed the promoter activity in differentiated F442-A adipocytes. Potential regulatory elements are discussed in this report.
Obesity is a common complex disease that increases the risk of hypertension and diabetes. Both genetic and environmental factors play a role in the development and maintenance of human obesity (for review, see (1, 2, 3, 4) ). The multifactorial nature of obesity makes the genetically obese animal a useful model to dissect the pathogenesis of the disease, and several different models of obesity have been established in rodents(5) . The mouse with the obese mutation (ob) is obese and develops diabetes, and is therefore a model for related human diseases(6) . Recently, the mouse ob gene and its human homolog were cloned; a premature stop codon mutation or the total absence of ob mRNA results in the obesity in the ob/ob mice. Structural analysis of the ob protein sequence suggests the protein may be secreted as a circulating factor to regulate body weight(6) .
The relationship of the ob gene product to human obesity is still not clear. No mutations were found in the coding region of human ob cDNA in obese people in a recent study(7) . The ob mRNA was found to be increased in ob/ob mice with a nonsense mutation (6) and in the obese people(7) , suggesting that the ob gene expression might be transcriptionally regulated. Thus, we are interested in how the ob gene expression is regulated in normal and obese subjects in order to understand the pathogenesis of obesity and the biology of the adipocyte. As a first step, we report here the isolation the human ob gene including its promoter region.
Figure 4: Nucleotide sequence of the human ob gene. The sequences of all the exons (uppercase letters), the promoter region, and exon/intron junction (lowercase letters for intron) are shown. Nucleotide numbering starts with the +1 as defined by primer extension and 5-RACE, and proceeds as indicated in the left margin, taking into account only exonic sequences. The deduced amino acid sequence and numbering are shown below the nucleotide sequence. The oligonucleotides used for PCR or probe are underlined. The putative TATA core promoter at -28 bp is bold, and potential transcription regulatory sites are double-underlined. C/EBP, CCAAT/enhancer-binding protein; GRE, glucocorticoid response element; CREB, cAMP response element-binding protein. The putative polyadenylation signal is boxed.
Human IMAGE consortium cDNA clones (LLNL) were obtained from Research Genetics, Inc.
Figure 1: Primer extension analysis on 5`-end of human ob gene. A, 5 µg of human fat total RNA (lanes 1 and 3) or yeast RNA (lanes 2 and 4) was subjected to primer extension with primers p166 (lanes 1 and 2) and p360 (lanes 3 and 4). The length of major extended products, determined by DNA ladders, is shown by arrows. B, schematic summary of the result with numbers referring to the start of exon 1.
Using end-labeled probes p143 and p148, the genomic fragments containing exon 2 and 3 were cloned, which encoded the Ob protein ( Fig. 2and Fig. 4). The sizes of the first and second introns were about 10 and 1.9 kb, as determined by restriction enzyme mapping and PCR, separately (Fig. 2; data not shown).
Figure 2: Genomic structure of the human ob gene. The position of the three exons are indicated by boxes. The coding region is depicted in black, while the untranslated regions are represented by open boxes. The restrictions sites used in subcloning and mapping are shown. The genomic fragments subcloned into pKS vectors are outlined in bold. An enlargement of the promoter region shows the relative positions of repetitive sequences and putative regulatory elements. The IMAGE consortium clones were aligned with exon 3.
The sequence analysis showed that the upstream sequence of
the genomic fragment C2012 included exon 3 and encoded the C terminus
of the Ob protein. A further analysis of the downstream sequence to
nucleotide 2241 revealed 71% identity to mouse 3`-UTR (Bestfit,
Genetics Computer Group, Inc.), indicating this region may be exonic.
3`-RACE was performed in order to isolate the entire human 3`-UTR. A
1.1-kb fragment was obtained after two-round PCR using primers first
p180 and nest p197 with an anchor primer (data not shown). Sequencing
revealed that the fragment was identical to the human genomic sequence
and shared homology to mouse ob cDNA, indicating the fragment
was a 3`-UTR of the human ob cDNA. The fragment apparently
ended at the poly(A) sequence corresponding to genomic DNA
(3066-3081), suggesting the ending might be a premature
termination due to the annealing of oligo(dT) in the
reverse transcription. A BLAST search (
)using the human ob 3`-sequence from positions 2541 to 3081 found perfect
matches to 5`-ends of two IMAGE consortium (LLNL) human cDNA
clones(10) . The clones were sequenced in full and found to be
identical to the human ob genomic sequence (Fig. 2).
Clones 139081 and 182874 were derived from a human placenta and breast
library, respectively, consistent with our Northern analysis that mRNA ob is expressed in human fat and placenta (data not shown).
Moreover, a putative termination signal AATAAA was found 20 bp before
the mRNA polyadenylation sequence in both clones, indicating they are
authentic 3`-ends of the human ob mRNA ( Fig. 2and 4).
BLAST search found that the 3`-UTR of the human ob mRNA
contains an Alu repetitive sequence from positions 3081 to 3370. The
physiological function of the repeat, if any, remains to be explored.
Taken together, the human ob gene was composed of three exons,
encoding a 3.5-kb cDNA and spanning around 18 kb of the genome ( Fig. 2and Fig. 4). The sequence of the intron/exon
junction followed the TG/GA rule for splicing.
Figure 3: Transfection assay of the human ob gene promoter. 2 µg of plasmid was transfected into differentiated F442A adipocyte in a six-well plate and assayed for luciferase activities. Deletions of the 5`-flanking region were constructed at the indicated restriction site. The vector pGL3-Basic has no promoter. Luciferase activities were normalized by protein content and are shown as percentages of that of pGL3/0.3kb(+) promoter. Each bar represents the mean and standard deviation of three independent transfections.
BLAST search with the 3-kb
promoter region reveals several features of the region. Two repetitive
sequences MER11 (medium reiteration frequency; (12) ) and Alu
were found from -2514 to -1545 and from -1364 to
-1046. Repetitive elements have been involved in regulation of
several genes, such as human CD8(13) , keratin 18 gene (14) , and immunoglobulin
light chain
gene(15) . Whether the above MER11 and Alu repeats are
functional in regulating the ob expression remains to be
studied.
A computer scan of the 3-kb 5`-flanking region with the TFD (Transcription factor data base, with 0 mismatch) from GenBank disclosed dozens of putative binding sites for known transcription factors. Of the notables are Sp-1 sites, cAMP response element, glucocorticoid response element, and CCAAT/enhancer-binding protein sites. The ectopic expression of CCAAT/enhancer-binding protein has been found to induce adipogenesis in F442A preadipocyte(16) . The ob gene expression has been reported to respond to insulin (11, 17) and dexamethasone(18) . Whether the above elements are involved in the regulation is under investigation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U43653 [GenBank]and U43589[GenBank].