The obese (ob) gene, an autosomal recessive
mutation on mouse chromosome 6, arose spontaneously in the mouse colony
at the Jackson Laboratory(1) . Mice homozygous for the ob mutation, known as ob/ob mice, develop severe hereditary
obesity and non-insulin-dependent diabetes mellitus. The molecular
identification of the ob gene by Friedman and co-workers (2) has provided new insight into the pathogenesis of obesity
and obesity-linked diabetes. The authors identified the mouse ob gene through a positional cloning strategy and determined the
structure of the mouse ob protein and also its human
homologue(2) . The ob protein, a 166/167-amino acid polypeptide
with a putative signal sequence, is highly conserved in structure among
species, and expression of the ob gene is abundant in and
specific to adipose tissue in mice(2) . Recently, we and others
have also 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) .
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 (fa/fa)(4, 6) and Wistar fatty (fa/fa) rats(8) ) and in rodent models of acquired
obesity obtained by pure overfeeding of normal rats (
)or by
ventromedial lesion to rat hypothalamus(7) . The augmentation
of ob gene expression in adipose tissue is also
region-specific(4, 7, 8) . Furthermore, ob gene expression is also 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. Indeed, nonsense mutation of the ob gene has been
proven to be the obesity-causing mutation in C57BL/6J ob/ob mice(2) . On the other hand, no such mutation of the ob gene has been found in human obesity(5) .
To
understand the physiologic and pathophysiologic roles of the ob gene in humans, it is important to elucidate the structural
organization of the human ob gene. Furthermore, molecular
characterization of the ob gene from any species has not so
far been reported. We report here the isolation and structural
organization of the human ob gene. Using the fluorescence in situ hybridization technique, we also determined the
chromosomal assignment of the human ob gene.
EXPERIMENTAL PROCEDURES
Genomic Southern Blot Analysis
Human genomic DNA
extracted from blood leukocytes was digested with restriction
endonucleases SacI, EcoRI, KpnI, SphI, and NcoI; electrophoresed on a 0.7% agarose gel
(5 µg/lane); and transferred onto a Biodyne A nylon membrane (Pall,
Glen Cove, NY)(9) . The membrane was prehybridized at 42 °C
in a solution containing 50 mM sodium phosphate buffer (pH
7.0), 5
SSC (1
SSC is 0.16 M NaCl and 0.016 M sodium citrate (pH 7.0)), 50% formamide, 5
Denhardt's solution, 0.1% SDS, and 200 µg/ml salmon testis
DNA. Hybridization was performed in the same solution plus the
P-labeled human ob cDNA fragment (3) as
a probe. The membrane was washed three times at 55 °C in 0.1
SSC and 0.1% SDS. The blot was used to expose an x-ray film
with an intensifying screen for 1 week.
Genomic Library Screening
A human genomic DNA
library derived from leukocyte DNA in
EMBL3 (CLONTECH, Mountain
View, CA) was screened with the
P-labeled human ob cDNA probe(3) . To obtain the 5`-flanking region of the
human ob gene, a second human genomic DNA library derived from
leukocyte DNA in
EMBL3 (CLONTECH) was screened with the
P-labeled human ob genomic fragment (fragment 1)
(see Fig. 2). Prehybridization and hybridization were carried
out as described(9, 10) . The membranes were washed in
2
SSC and 0.1% SDS twice at 60 °C and in 0.2
SSC
and 0.1% SDS three times at 60 °C. Appropriate restriction
fragments were subcloned into the pBluescript vector (Stratagene Inc.,
La Jolla, CA) for further analysis.
Figure 2:
Schematic representation of the structure
of the human ob gene and cDNA. a, the genomic clones
(
OB3-1,
OB1-8, and fragment 1) and the BamHI (B) and XhoI (X) restriction sites. b, the sequencing strategy. Arrows denote the extent
of sequence obtained. c, the structure of the human ob gene. Exons are boxed and numbered. The coding
region is indicated by closed boxes. d, the structure
of human ob cDNA. The coding region is depicted by a closed box. The translation start (ATG) and stop (TGA) sites
are indicated.
Polymerase Chain Reaction
PCR (
)was
used to obtain the genomic fragment that contains the first exon and
the upstream half of the first intron of the human ob gene
(fragment 1) (see Fig. 2). Using a Model 381A DNA synthesizer
(Applied Biosystems Inc., Foster City, CA), two oligonucleotide primers
(sense, 5`-TAGGAATCGCAGCGCCAACGGTT-3`; antisense,
5`-CTACTTGGGAGGCCAAGGTGGGAGGTTTGC-3`) were synthesized based upon the
nucleotide sequences of human ob cDNA (3) and the
first intron of the human ob gene, respectively (see Fig. 2). Using human genomic DNA as template, PCR was performed
with a Takara Shuzo LA PCR kit. The reaction profile was as follows:
denaturation at 98 °C for 20 s and annealing and extension at 68
°C for 3 min for 30 cycles. The amplified DNA fragment of 5.3 kb in
size was subcloned into the pGEM-T vector (Promega, Madison, WI) for
sequencing.
Tissue Preparation and RNA Extraction
Human
adipose tissue was obtained at the time of operation from the
subcutaneous abdominal fat pad of a 58-year-old female patient with
gastric cancer. Tissues were frozen in liquid nitrogen and stored at
-70 °C until use. Total RNA extraction was carried out as
described(3, 4, 8) .
Reverse Transcription-PCR
RT-PCR was performed to
determine the presence or absence of any introns in the 3`-untranslated
region of the human ob gene. Approximately 10 µg of total
RNA from human adipose tissue was reverse-transcribed by random hexamer
priming using Superscript Moloney murine leukemia virus reverse
transcriptase (Life Technologies, Inc.). The single-stranded cDNA was
subjected to PCR as described(11) . The human ob cDNA-specific PCR primers were generated using a Model 381A DNA
synthesizer. Amplified DNA fragments were subcloned into the pGEM-T
vector for sequencing. The presence or absence of any introns in the
3`-untranslated region was determined by comparison of the nucleotide
sequences of the cloned human ob genomic fragment with those
of the RT-PCR products that cover the entire 3`-untranslated region of
the human ob gene.
Rapid Amplification of 5`-cDNA Ends (5`-RACE)
The
5`-RACE experiment was performed essentially as described (4) using the 5`-AmpliFINDER
RACE kit (CLONTECH).
Approximately 10 µg of total RNA from human adipose tissue was
reverse-transcribed by a human ob cDNA-specific antisense
primer (5`-ATGGGGTGGAGCCCAGGAAT-3`). The single-stranded cDNA was
ligated to the AmpliFINDER anchor and amplified by PCR using the
AmpliFINDER anchor primer and a second upstream human ob cDNA-specific antisense primer (5`-TTGGATGGGCACAGCTTG-3`). A
single fragment of
200 bp in size was obtained, which was
subcloned into the pGEM-T vector for sequencing.
Rapid Amplification of 3`-cDNA Ends (3`-RACE)
The
3`-RACE experiment was carried out as described (9) to
determine the 3`-end of the human ob gene. Approximately 10
µg of total RNA from human adipose tissue was reverse-transcribed
by adaptor oligo(dT)
priming
(5`-GGCAGTCCGAATTCCTCGAGTTTTTTTTTTTTTTT-3`) using Superscript Moloney
murine leukemia virus reverse transcriptase. After synthesis of the
second strand cDNA by a 5`-gene-specific primer
(5`-GGCCAGAAGAATTGAGATTC-3`), PCR was carried out using the primer and
the adaptor oligonucleotide (without 13 dT nucleotides on the 3`-end) (7) . An aliquot of the reaction was further subjected to PCR
using a downstream 5`-gene-specific primer (5`-TAGGCTGAGGCAGGAGAATC-3`)
and the adaptor primer. The 3`-RACE product was analyzed by a 1.5%
agarose gel, and amplified DNA was subcloned into the pGEM-T vector for
sequencing.
DNA Sequencing
Nucleotide sequences were
determined by the dideoxy chain termination method (12) using
Sequenase version 2.0 (U. S. Biochemical Corp.) and a DyeDeoxy
Terminator Cycle sequencing kit (Applied Biosystems Inc.).
Sequence-specific primers were synthesized using a Model 381A DNA
synthesizer. All DNA sequences were confirmed by reading both DNA
strands.
Fluorescence In Situ Hybridization
Metaphase
spreads were prepared from phytohemagglutinin-stimulated lymphocyte
culture by a thymidine synchronization, 5-bromodeoxyuridine release
technique for the delineation of G-bands. Before hybridization in
situ, chromosomes were stained in Hoechst 33258 and irradiated
with UV(13) . The BamHI/SalI-digested
fragments of the isolated genomic clone (
OB1-8) (see Fig. 2) were labeled with biotin-16-dUTP (Boehringer Mannheim
GmbH, Mannheim, Germany) by nick translation. Hybridization signals
were detected with fluorescein isothiocyanate-avidin (Boehringer
Mannheim GmbH), and chromosomes were counterstained with propidium
iodide (1 µg/ml). The precise signal positions were determined by
the delineation of G-band patterns as
described(14, 15) . Microscopy was performed with a
Nikon FXA fluorescent microscope. Propidium iodide-stained chromosomes
and fluorescein isothiocyanate signals were visualized through a Nikon
B-2A filter, and G-band patterns on the same metaphase chromosomes were
delineated through a Nikon UV-2A filter.
RESULTS
Genomic Southern Blot Analysis
Southern blot
analysis of human genomic DNA with the human ob cDNA probe
identified a single hybridizing band upon digestion with restriction
endonucleases EcoRI, KpnI, and SphI (3.7,
18, and 4.3 kb in size, respectively). On the other hand, digestion
with SacI and NcoI gave two hybridizing bands of 7.2
and 3.8 kb in size and of 5.8 and 5.1 kb in size, respectively (Fig. 1).
Figure 1:
Southern blot analysis of human genomic
DNA. Samples of human genomic DNA (
5 µg/lane) digested with SacI, EcoRI, KpnI, SphI, and NcoI were analyzed by 0.7% agarose gel electrophoresis,
blotted, and hybridized with the
P-labeled human ob cDNA probe(3) . The HindIII fragments of
-DNA were used as size markers.
Isolation and Characterization of the Human ob Genomic
Fragments
To isolate the human ob gene,
6
10
recombinants from a human genomic DNA library in
EMBL3 were screened with the
P-labeled human ob cDNA probe(3) . A single positive clone (
OB1-8)
harbored an
14-kb human ob genomic fragment, which
contained the 5.3-kb downstream half of the first intron and the second
and third exons of the human ob gene (Fig. 2). The
5.3-kb genomic fragment (fragment 1) was amplified by PCR and contained
the first exon (
29 bp) and the 5.3-kb upstream half of the first
intron (Fig. 2). To obtain the 5`-flanking region of the human ob gene,
5
10
clones from a second
human genomic DNA library in
EMBL3 were screened with
P-labeled fragment 1. Six positive clones were identified
and plaque-purified. DNA from one clone (
OB3-1) harbored an
16-kb genomic DNA fragment that contained the 5.0-kb 5`-flanking
region of the human ob gene (Fig. 2).
Structural Organization of the Human ob Gene
Fig. 3shows the nucleotide and deduced amino acid
sequences of the human ob gene. The exon/intron borders were
determined by comparison of the nucleotide sequences of the human ob gene with those of human ob cDNA(3) . The
human ob gene spanned
20 kb and was organized into three
exons separated by two introns. Splicing donor and acceptor consensus
sequences (16) were located at the putative exon/intron
borders. The first intron was
10.6 kb in size and occurred in the
5`-untranslated region, 29 bp upstream of the ATG start codon. The
second intron,
2.3 kb in size, was located at glutamine +49.
The third exon contained the downstream coding region and the
3`-untranslated region of the human ob gene. Since complete
nucleotide sequences of the 3`-untranslated region of the human ob cDNA have not yet been reported, nucleotide sequences of the third
exon were determined by sequencing the 3`-RACE product and the
corresponding genomic regions. Comparisons of the nucleotide sequences
of the human ob genomic regions with those of the
3`-RACE/RT-PCR products that cover the entire 3`-untranslated region
revealed the absence of any introns in the 3`-untranslated region of
the human ob gene (data not shown).
Figure 3:
Nucleotide and deduced amino acid
sequences of the human ob gene. Exon sequences are shown in upper-case letters. Introns and putative 5`- and 3`-flanking
sequences are shown in lower-case letters. Nucleotides are numbered, with position +1 referring to the first
nucleotide of the ATG start codon. The transcription initiation sites
are marked by inverted triangles. The translation stop codon
is marked by triple asterisks. The GT-AG sequences found at
the splice sites are underlined. The CT-rich sequence in the
3`-flanking region is indicated by dashed lines. The 5`- and
3`-ends of the cloned human ob cDNA (3) are indicated
by closed circles. The deduced amino acid sequence is shown in three-letter code above the nucleotide sequence. Amino acids
are also numbered sequentially from the translation start
site.
Determination of the Transcription Initiation Sites of
the Human ob Gene
To determine the transcription initiation
sites of the human ob gene, the 5`-RACE experiment was carried
out. To exclude the nucleotide misincorporation during the PCR
amplification, a total of 10 clones were sequenced. Sequence analysis
identified the transcription initiation sites 54
57 bp upstream of
the ATG start codon (G at position -57, three clones; T at
position -56, one clone; A at position -55, one clone; G at
position -54, five clones) (Fig. 3). The 5`-end of the
cloned human ob cDNA (3) was located 44
47 bp
downstream of the transcription initiation sites (Fig. 3). The
5`-ends of mouse and rat ob cDNAs have been located 57 and 60
bp upstream of the ATG start codon, respectively (2, 4, 17) . Although there is a high
nucleotide sequence similarity in the 5`-untranslated region between
mouse and rat ob cDNAs (93%), nucleotide sequences of the
5`-untranslated region of the human ob gene were less
homologous to those of mouse and rat ob cDNAs (51 and 47%,
respectively).
Analysis of the 5`-Flanking Region of the Human ob
Gene
The 172-bp 5`-flanking region of the human ob gene
sequenced in this study contained a TATA box-like sequence (TATAWAW, W
= A/T; positions -87 to -81) (16) 27
30
bp upstream of the transcription initiation sites (Fig. 4). A
computer search of the 5`-flanking region for cis-acting
regulatory elements also revealed the presence of three copies of GC
boxes (GGGCGG) (18) at positions -79 to -74,
-155 to -150, and -160 to -155; a binding site
for CCAAT/enhancer-binding protein (C/EBP) (TKNNGYAAK, K = G/T,
N = A/C/G/T, and Y = C/T) (19) at positions
-111 to -103; an E box (CANNTG, N = A/C/G/T) (20) at positions -114 to -109; and an AP-2-binding
site (CCCAGGGC) (21) at positions -199 to -192.
Figure 4:
Nucleotide sequence of the 5`-flanking
region of the human ob gene. The transcription initiation
sites are indicated by inverted triangles. A TATA box-like
sequence, three copies of GC boxes, an E box, a C/EBP-binding site, and
an AP-2-binding site are boxed.
Analysis of the 3`-Flanking Region of the Human ob
Gene
Molecular cloning studies of mouse, rat, and human ob cDNAs(2, 3, 4, 5, 6, 7) have
revealed no polyadenylation sites for their mRNAs, and the 3`-end of ob cDNA from any species has never been elucidated. To
determine the 3`-end of human ob cDNA, the 3`-RACE experiment
was carried out. Using total RNA from human adipose tissue, a single
band of
130 bp in size was obtained. Sequence analysis of the
3`-RACE products revealed that the cytosine nucleotide at position
+4183 is followed by the poly(A) stretch (data not shown),
suggesting that the cytosine nucleotide at position +4183 is the
3`-end of human ob cDNA or the 3`-end of the third exon of the
human ob gene (Fig. 3). The overall size of the three
exons (4240 bp) and the potential poly(A) stretch (usually
200 bp)
is consistent with that of human ob mRNA (
4.5 kb) as
revealed by Northern blot analysis(3) . No typical
polyadenylation signal (AATAAA) (22) was found near the
putative poly(A) addition site. Nucleotide sequences of the
3`-untranslated region of the human ob gene were
50%
homologous to those of mouse ob cDNA(2) . In the
3`-flanking region of the human ob gene, there was a
characteristic CT-rich sequence at positions +4417 to +4538 (Fig. 3).
Chromosomal Assignment of the Human ob Gene
The
chromosomal localization of the human ob gene was determined
by the fluorescence in situ hybridization technique (Fig. 5). A total of 50 metaphase cells were examined. Of these,
nine cells (14%) exhibited twin-spot signals on both homologous 7q31.3
chromosomes, and the other 17 cells (34%) had twin-spot signals on one
7q31.3 chromosome and a single spot on another 7q31.3 chromosome. Such
specific accumulation of the signals could not be detected on any other
chromosomes. These results indicate that the human ob gene is
localized on chromosome 7q31.3.
Figure 5:
Chromosomal assignment of the human ob gene by the fluorescence in situ hybridization technique. Left, partial metaphase chromosomes stained with propidium
iodide showing the twin-spot signals on the long arms of both
homologous chromosomes 7 (arrowheads); right, the
G-band pattern of the same chromosomes delineated through a Nikon UV-2A
filter. These results clearly indicate that the human ob gene
is localized on the region of chromosome
7q31.3.
DISCUSSION
In this study, we succeeded in the isolation and
characterization of the human ob gene. Southern blot analysis
of human genomic DNA identified a single hybridizing band upon
digestion with EcoRI, KpnI, or SphI and two
hybridizing bands upon digestion with SacI or NcoI (Fig. 1). These results are consistent with the restriction
endonuclease map showing that a single site of SacI and NcoI is observed in the second intron of the genomic region
that covers the ob cDNA sequence used as a probe, while EcoRI, KpnI, and SphI sites are not present
(data not shown). These results indicate that the ob gene is
present as a single-copy gene in the human genome. Using the mouse ob cDNA fragment as a probe, Zhang et al.(2) identified, by Southern blot analysis of human genomic
DNA, a single hybridizing band of >11 kb in size upon digestion with EcoRI. Differences in the size of the hybridizing band
observed may represent the restriction fragment length polymorphisms
between the human genomic DNAs used.
This study demonstrates that
the human ob gene is composed of three exons separated by two
introns. The first intron occurred in the 5`-untranslated region, and
the coding region was separated by a single intron at glutamine
+49. It has been demonstrated that in mice and humans, two
different cDNAs encode the 166/167-amino acid ob proteins, which differ
in the presence or absence of glutamine
+49(2, 3) . On close inspection, there are one
donor and two acceptor sites (18) around the junction region (Fig. 3). Furthermore, we have also observed that there is an
internal alternative splice site (23) at glutamine +49 of
the mouse ob gene. (
)These observations suggest
that the two ob proteins in mice and humans are generated by the
alternative mRNA splicing mechanism.
The 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, a
C/EBP-binding site, an E box, and an AP-2-binding site). The C/EBP
transcription factor has been implicated in the coordinate
transcriptional activation of adipocyte-specific genes during the
course of adipocyte differentiation(24, 25) . We (
)and others (26) have observed that ob gene expression is induced in stromal-vascular cells or 3T3-F442A
preadipocytes during the course of adipocyte development and/or
maturation, although no significant amount of ob mRNA is
present in undifferentiated cells. Therefore, the C/EBP-binding site in
the 5`-flanking region of the human ob gene might be involved
in the transcriptional activation of the ob gene during
adipocyte differentiation. Further studies are needed to elucidate the
functional significance of these cis-acting regulatory
elements.
We demonstrated by the fluorescence in situ hybridization technique that the ob gene is mapped on
human chromosome 7q31.3. Of particular note is that the cystic fibrosis
transmembrane conductance regulator gene has been assigned to human
chromosome 7q31.3 (27) . It has been demonstrated that the ob gene is localized on the proximal region of mouse
chromosome 6(2, 28) . The mouse chromosomal region on
which the ob gene is located is part of a known segment with
genes that are conserved between mice and humans and is syntenic to
human chromosome 7q(29) . This study has provided direct
evidence that the ob gene is a member of the conserved
syntenic group in mice and humans and further helps gene mapping in
both species.
In conclusion, we succeeded in the isolation and
characterization of the human ob gene. Using the fluorescence in situ hybridization technique, we also determined the
chromosomal assignment of the human ob gene. This study helps
to establish the genetic basis of ob gene research in humans,
thereby leading to a better understanding of the physiologic and
pathophysiologic implications of the ob gene.