From the Department of Biochemistry, Dartmouth
Medical School, Hanover, New Hampshire 03755, § Shanghai
Institute of Biochemistry, Shanghai, China,
Departments of Cell Biology and Medicine, Baylor
College of Medicine, Houston, Texas 77030, and the
** Department of Pathology, Dartmouth-Hitchcock Medical
Center, Lebanon, New Hampshire 03756
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ABSTRACT |
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Acyl-CoA:cholesterol acyltransferase (ACAT) plays
important roles in cellular cholesterol homeostasis. Four human ACAT-1
mRNAs (7.0, 4.3, 3.6, and 2.8 kilobases (kb)) share the same short
5'-untranslated region (exon 1) and coding sequence (exons 2-15). The
4.3-kb mRNA contains an additional 5'-untranslated region (1289 nucleotides in length; exons Xa and Xb)
immediately upstream from the exon 1 sequence. One ACAT-1 genomic DNA
insert covers exons 1-16 and a promoter (the P1 promoter). A separate
insert covers exon Xa (1277 base pairs) and a different
promoter (the P7 promoter). Gene mapping shows that exons 1-16 and the
P1 promoter sequences are located in chromosome 1, while exon
Xa and the P7 promoter sequence are located in chromosome
7. RNase protection assays demonstrate three different protected
fragments, corresponding to the 4.3-kb mRNA and the two other
mRNAs transcribed from the two promoters. These results are
consistent with the interpretation that the 4.3-kb mRNA is produced
from two different chromosomes, by a novel RNA recombination mechanism
involving trans-splicing of two discontinuous precursor RNAs.
Acyl-CoA:cholesterol acyltransferase
(ACAT)1 is an intracellular
enzyme present in a variety of human and other animal tissues. It
catalyzes the formation of cholesteryl esters from cholesterol and
long-chain fatty acyl coenzyme A (for a review, see Ref. 1). In
addition to storing cholesterol intracellularly, ACAT plays important
physiological roles, including hepatic lipoprotein assembly, dietary
cholesterol absorption, and steroidogenesis (for reviews, see Refs.
2-4). Under pathological conditions, accumulation of cholesteryl
esters produced by ACAT is characteristic of foam cell formation in
atherosclerotic lesions (for a review, see Ref. 5; also see Ref. 6).
For these reasons, ACAT has been a pharmaceutical target for developing
cholesterol-lowering and/or anti-atherosclerosis agents (for a review,
see Ref. 7). The human ACAT-1 cDNA was cloned by a somatic cell and
molecular genetic approach. Chinese hamster ovary (CHO) cell mutants
lacking ACAT activity (including clone AC29) were isolated (8);
subsequent stable transfection experiments showed that human genomic
DNAs complemented the ACAT deficiency in AC29 cells (9). A 1.2-kb human
genomic DNA fragment was cloned from the stable transfectants. This
fragment (designated as G2 DNA) led to the eventual cloning of a
full-length human ACAT cDNA K1 (4011 bp in length). Expression of
this cDNA, designated as ACAT-1, in AC29 cells complemented the
ACAT deficiency of the mutant (10). Additional results showed that
expressing this cDNA in insect cells, which do not contain endogenous ACAT-like activity, produced high levels of ACAT activity in vitro, confirming that this cDNA encodes the
catalytic component of ACAT enzyme (11). The coding region of the ACAT
gene has been mapped to chromosome 1q 25 (12). Protein sequence
analysis revealed the ACAT-1 protein as a hydrophobic protein
containing multiple transmembrane domains and sharing several peptide
regions in common with other acyltransferases (10). Recombinant human ACAT-1 protein expressed in CHO cells has been purified to homogeneity; the homogeneous ACAT-1 protein remains catalytically active and uses
cholesterol as a substrate in a highly cooperative manner (13).
Homologues of human ACAT-1 cDNA have also been cloned from other
species (reviewed in Ref. 1), including two yeast homologues (14, 15).
Disruption of the ACAT-1 gene in mice has been reported (16); the
ACAT-1 gene-deficient mice exhibit marked reduction in cholesteryl
ester levels in only selective tissues and not in all the tissues
examined. These and other results led to the molecular cloning of
ACAT-2 cDNA (17-19). The predicted amino acid sequence of ACAT-2
is homologous but distinct from that of ACAT-1. The physiological roles
of ACAT-1 and ACAT-2 in various tissues of different species are
currently under intense investigation by several laboratories. In
humans, immunodepletion experiments suggest that the ACAT-1 protein
plays major catalytic roles in hepatocytes, adrenal glands,
macrophages, and kidneys, but not in the intestines (20).
The 4.0-kb human ACAT-1 cDNA contains a single open reading frame
of 1.65 kb. It also contains an unusually long 5'-untranslated region
(5'-UTR; 1396 bp) and 965 bp of 3'- untranslated region. Using the
coding region as probe, Northern analyses have revealed the ubiquitous
presence of four ACAT-1 mRNAs (with sizes estimated at 7.0, 4.3, 3.6, and 2.8 kb) in all of the human tissues and various human cell
lines examined (10, 21-23). Four different ACAT mRNAs have also
been detected in all of the tissues isolated from rabbits (21). The
origins and the relationship of these mRNAs are not clear. Using
specific antibodies against the predicted human ACAT-1 protein sequence
as the probe, only one major protein band, with an apparent molecular
mass of about 50 kDa, has been detected with certainty in extracts of
various human cells and tissues by SDS-PAGE analysis (20, 24).
Experiments performed in various cultured mammalian cells have
indicated that the main mode of regulation of ACAT-1 by sterol is at
the post-translational level (22, 24). An allosteric regulation model
that involves activation of ACAT-1 enzyme through configurational
change(s) of ACAT-1 protein upon binding to cholesterol/oxysterol has
been proposed (11, 13; reviewed in Ref. 1). Additional regulatory mechanisms have also been suggested: experiments in rabbits and mice
have demonstrated that ACAT-1 mRNAs undergo tissue-specific increases upon feeding the animals with an atherogenic diet rich in fat
and cholesterol (21, 25). ACAT-1 mRNAs and protein content are
significantly up-regulated during the human monocyte-macrophage differentiation process in vitro (6, 23); in mouse
macrophages, dexamethasone is shown to up-regulate ACAT-1 mRNAs
(26).
To provide the molecular basis for further understanding the regulation
of ACAT protein and ACAT gene, we now report the genomic organization
of the human ACAT-1 gene, including the identification and
characterization of two promoter regions. We also report the surprising
finding that the ACAT-1 P1 promoter region and the exon 1-16 sequences
are located on chromosome 1(1q 25), while the ACAT-1 P7 promoter region
and the optional exonic sequence (exon Xa) are located on
chromosome 7 (7q 31.3). The results show that one of the ACAT-1
mRNAs, the 4.3-kb ACAT-1 mRNA, is produced from two different chromosomes.
Isolation and Characterization of the Human ACAT Genomic
DNAs--
A 1.2-kb human ACAT-1 genomic DNA fragment (designated as G2
DNA), known to contain exonic human ACAT-1 sequences, has previously been isolated from CHO cell transfectants that stably express human
ACAT activity (10). DNA sequencing reveals that G2 DNA contained 1139 bp. The sequence includes the 79-bp exon 8, a 668-bp intron 8, an 82-bp
exon 9, and a 310-bp piece of intron 9 (result not shown). Within
intron 8, a pair of oligomers has been found to give rise to a single
411-bp PCR product when either the G2 DNA or the total human genomic
DNA was used as the template. The sequence for the forward primer is
5'-GGCTGGATATTTCACCCTTTG-3'; the sequence for the reverse primer is
5'-GAATATAAGGAAGCACAGAAC-3'. This pair of oligomers was sent to Genomic
Systems, Inc. (St. Louis, MO) for PCR screening the human genomic DNA
P1 library (human DNA fragments cloned in the BamHI site of
P1 vector pAD10SacB II). Three positive P1 clones have been isolated
(P1 733, 734, and 774). Detailed analyses revealed that these
overlapping P1 clones contain the human ACAT-1 protein coding regions,
but they did not contain the unusually long 1289-bp region located at
the 5'-UTR of human ACAT cDNA K1. To isolate additional P1 clone(s) that cover this region, two additional primers were selected from the
5'-UTR of human ACAT cDNA K1; the sequence for the forward primer
is 5'-GCACGGGTTAAGATCT-3' (human ACAT cDNA K1 nt 982-997), and the
sequence for the reverse primer is 5'-GATAACCCACTGGAAG-3' (human ACAT
cDNA K1 nt 1181-1196). Using the second pair of primers, further
screening (done at Genomic Systems) yielded a single P1 clone (P1
4651). The P1 clones were restriction-mapped by digestion with a
combination of restriction endonucleases, electrophoresed on 1%
agarose gels, and stained by ethidium bromide. The sizes of digested
fragments were determined by comparison with DNA size standards from
Life Technologies, Inc. The sizes of larger fragments (larger than 4 kb) were confirmed by estimating the sizes of their composite fragments
after subcloning and further digestion. Appropriate restriction
fragments were subcloned into linker sites of pBluescript vectors
(Stratagene) for sequencing. DNA sequencing was performed using P1
clones and/or their subcloned fragments as the templates. Sequencing
was according to the double-stranded DNA cycle sequencing system kit,
and Tag DNA polymerase was supplied by Life Technologies, Inc.
Exon/intron junctions were determined by using sequencing primers
corresponding to nucleotide sequences of the human ACAT cDNA K1
(10).
Determination of the 5'-Ends of Human ACAT-1
mRNAs--
Rapid amplification of the 5' cDNA ends (5'-RACE)
was performed using the 5'-SLIC (single strand ligation to single
strand cDNA) strategy (27, 28). Total RNAs used as templates were prepared from HeLa cells, human U937 cells, or various native human
tissues as indicated, with TRIzol reagent (Life Technologies, Inc.).
First strand cDNA synthesis was performed using SUPERScript II
reverse transcriptase. The reaction conditions were as described in the
instruction manual provided by Life Technologies, Inc.; the primer used
was ACAT specific reverse primer 5'-ACCCACCATTATCTAA-3', located at
human ACAT cDNA K1 nt 1670-1655. The conditions for ligating the
oligonucleotide to the single strand cDNA were set according to the
protocol described (28). The sequence of the anchor oligonucleotide
used was a 28-mer: 5'-CGTCGACTATAGAGCGGCCGCAAGCTTT-3'. After the
ligation, PCR amplification was performed with 2-µl aliquots of the
terminated ligation mixtures. For PCR, the primer specific for the
anchor primer was 5'-AAAGCTTGCGGCCGCTCTATAGTCGACG-3'; the primer
specific for the human ACAT-1 cDNA was the reverse primer
5'-CGGCAGCGGGCACTTC-3' (designated as ACAT22; located at human ACAT K1
cDNA nt 1374-1359). The reactions were run as follows: cycle 1, 5 min at 94°; cycles 2-32, 45 s at 94°, 45 s at 54°, and
90 s at 70°. To analyze the size of the PCR products, a 20-µl aliquot of the PCR reaction product was electrophoresed through a 1.2%
agarose gel and stained with ethidium bromide; RNase Protection Assay--
For the RNase protection assay, a
365-nt radiolabeled antisense riboprobe was synthesized by in
vitro transcription of the cloned human ACAT cDNA fragment (nt
1113-1374, encompassing part of exon Xa, all of exon
Xb, and part of exon 1) in the presence of
[ Primer Extension Analysis--
Primer extension experiments were
carried out using SuperscriptTMII RNase H-
reverse transcriptase from Life Technologies, Inc. In accordance with
the instruction manual, 1 pmol of 5'-end-labeled human ACAT-1 specific
reverse primer (5'-ATCCCAGCACTTTAGGAGGCCG-3'; human ACAT cDNA K1 nt
92-71) was hybridized to 2 µg of human liver poly(A)+
RNA at 70 °C for 10 min. The reverse transcription reaction was performed at 42 °C for 50 min. The product was analyzed with a sequencing gel.
Luciferase Activity Assays Using the P1 or P7 Promoter of the
Human ACAT-1 Gene--
Various fragments within the Northern Analysis--
Poly(A)+ RNAs were prepared
from human melanoma A2058 cells using the FastTrack kit (Invitrogen).
Riboprobes were used for hybridization. To prepare the riboprobes,
various indicated regions of human ACAT cDNA K1 were subcloned into
pBluescript(SK Analysis of the (GT)n Microsatellite within the Human
ACAT Gene--
To examine the polymorphism of this repeat sequence,
genomic DNAs from 372 individuals were used to perform PCR using two primers flanking the GT repeat region. The DNA samples were obtained from Professor Eric Boerwinkle at the Human Genetics Center, University of Texas Health Science Center, Houston, TX. The two primers used are
indicated in Fig. 12 (boldface and italic
sequences). Briefly, approximately 200 ng of genomic DNA was
used in 20 µl of a PCR volume; the reaction mixture contained 1 mM dNTP, 1.25 mM forward primer, 1.25 mM reverse primer, and 1.5 mM magnesium
chloride. Thirty cycles under the following conditions were used for
the PCR amplification. Template DNA was denatured at 92° for 90 s, followed by annealing at 55° for 30 s and extension at 72°
for 30 s. The reverse primer used in the PCR was labeled with
[32P]ATP to detect the amplification product on a
sequencing gel. PCR products were run on a 6% polyacylamide, 8 M urea gel along with a sequencing ladder for size
determination. The major allele containing 14 GT repeats was confirmed
by sequencing.
Gene Mapping Studies--
To perform fluorescence in
situ hybridization (FISH) experiments, the genomic DNA inserts in
P1 4651 and P1 774 were biotin-labeled by nick translation (Bionick;
Life Technologies, Inc.). The hybridization solution contained 0.2 µg
of labeled probe, 10 µg of Cot-1 DNA (Life Technologies), and 30 µg
of herring sperm DNA (Life Technologies, Inc.) in 15 µl of Hybrisol
VII (Oncor)/slide. The probe mixture was heat-denatured at 70 °C for
5 min and allowed to preanneal at 37 °C for 2 h. Chromosome
preparations on slides were conditioned prior to hybridization in a
30-min, 37 °C bath in 2× SSC, followed immediately by dehydration
at room temperature in 70, 80, and 95% EtOH (2 min each), and
air-dried. The slides were then denatured in 70% formamide, 2× SSC at
70 °C for 5 min. followed by serial dehydration at room temperature.
Hybridization was carried out for 18 h in a moist 37 °C
chamber. Slides were washed in 50% formamide, 2× SSC at 37 °C for
30 min, followed by 2× SSC at 37 °C for 10 min. Slides were further
washed three times at room temperature in phosphate-buffered detergent
prior to signal detection. Hybridized DNA was detected with
avidin-fluorescein isothiocyanate, followed by a single round of
amplification according to the supplier's instructions (Oncor). FISH
signals were captured using a monochromatic CCD camera mounted on a
Zeiss epifluorescence microscope with a LUDL filter wheel and a fixed
multi-band pass beam splitter using MacProbe software (PSI, Houston,
TX). The P1 774 insert, containing the exon 1-15 region and 3'-UTR of
the human ACAT-1 gene, was mapped to chromosome 1q 25 (result not
shown), confirming our early report with the cDNA fragment as probe
(12). Using the P1 4651 insert as probe, analysis of 25 metaphases
showed that 14 had four signals, nine had three signals, and two had two signals at 7q31.3. No background signals (sites with >2 signals) were observed. Four signals are expected in a metaphase under conditions of fully efficient hybridization and signal detection, representing four copies of the locus, two on each of the replicated homologues. These results allow localization of the human ACAT-1 exon
Xa region to band 7q31.3.
The DNA sources for PCR analyses shown in Fig. 9 were genomic DNAs
prepared from human fibroblasts, mouse A9 cells, mouse A9 cells
containing human chromosome 1 (A9-1), or mouse A9 cells containing
human chromosome 7 (A9-7). The mouse A9 cells containing human
chromosome 1 or chromosome 7 were purchased from the Coriell Institute
for Medical Research (Camden, NJ). The details for conducting the PCR
described in Fig. 11 are described in the figure legend.
Other Methods--
Standard molecular biology techniques were
performed according to methods described by Sambrook et al.
(32). Adult and fetal human tissues were obtained from national
hospitals in Shanghai, China. Consents were obtained from donors or
their relatives for removal of tissues for research purposes. All human
tissues were of donor transplantation quality. The tissues were rapidly
frozen and stored in liquid nitrogen until use. For preparation of
poly(A)+ RNAs serving as templates for RT-PCR experiments
described in Fig. 1, the total RNAs from
the indicated human tissues were prepared using guanidinium thiocyanate
extraction followed by centrifugation in cesium chloride solution (32).
The poly(A)+ RNAs were isolated from the total RNAs by the
oligo(dT)-cellulose affinity column chromatography procedure (32). CHO
cells, human breast cancer cells, melanoma cells, and A293 cells were
grown in appropriate media as described previously (24). Stable
transfectants of CHO cells were selected by growing cells in G418
according to procedures previously described (10). Cloning of
individual transfectants with cloning rings was carried out according
to procedures previously described (10). For measurement of cholesteryl ester biosynthesis in intact cells, the [3H]oleate
pulse in intact cells followed by lipid analysis was according to
procedures previously described (33).
Presence of the Long 5'-UTR in Human ACAT-1 mRNA--
The
human ACAT cDNA K1 contains an unusually long 5'-UTR (1396 bp).
Within this region, an EcoRI site (nt 1282-1287) is
present. To rule out the possibility that nucleotides 1-1289 of human
ACAT cDNA K1 may be a ligation artifact produced during cDNA
synthesis in vitro, RT-PCR experiments were performed using
poly(A)+ RNAs isolated from adult human brain, intestine,
or liver as templates. The oligonucleotides relative to human ACAT
cDNA K1 nt 982-997 served as the forward primer and nt 1395-1410
as the reverse primer. These two primers are located in regions
flanking the EcoRI site described above. With these
templates, a single 429-bp PCR product was obtained (Fig.
1A). Direct sequencing of the PCR product revealed the
sequence to be identical to that found in human ACAT cDNA K1. Part
of the PCR product sequence is shown in Fig. 1B. Additional
experiments gave the same results when poly(A)+ RNAs from
human liver tumor Hep G-2 cells or human monocytic THP-1 cells were
used as the templates. Also, numerous control experiments showed that
when poly(A)+ RNAs from mouse liver or yeast tRNA was used
as template, no PCR products of discrete size were detected. The
positive results and the negative control experiments described above
have been consistently reproduced in T. Y. Chang's laboratory
(Hanover, NH) and B. L. Li's laboratory (Shanghai, China).
Together, these results indicate that the long 5'-UTR present in human
ACAT cDNA K1 is a genuine exonic region contiguous with the human
ACAT-1 coding sequence present in an authentic ACAT-1 mRNA.
Multiple Human ACAT-1 mRNAs--
Using the entire human
ACAT-1 coding region as probe, previous experiments have suggested the
existence of multiple human ACAT-1 mRNAs (with sizes of
approximately 7.0, 4.3, 3.6, and 2.8 kb). To examine the origin of
these mRNAs, we prepared various riboprobes derived from small
segments of the human ACAT cDNA K1, covering various parts of the
5'-untranslated, coding, and 3'-untranslated regions (lengths and
locations of these probes are indicated in the Fig.
2 legend). These probes were labeled and
hybridized individually with poly(A)+ RNAs from human
melanoma cells. The results (Fig. 2) show that the short 5'-UTR
sequence (exon 1; recognized by probe 5'U1), the various
coding regions (recognized by probes Cod1 to -4), and the proximal
portion of the 3'-untranslated region (recognized by probe 3' U1) were
present in all four types of human ACAT-1 mRNAs. In contrast, the
long 5'-UTR (recognized by probes 5'U2 and
5'U3) of human ACAT cDNA K1 is present only in the
4.3-kb human ACAT-1 mRNA. Lane 4 of Fig. 2 also shows that probes 5'U2 and
5'U3 cross-hybridized with an additional 2.3-kb RNA
species. Since this species was not recognized by probes within the
human ACAT-1 coding region, the significance of this observation is
unknown at present.
Cloning the Genomic DNA of the Human ACAT-1 Gene--
The human
genomic DNA inserts in four P1 clones (P1 733, 734, 774, and 4651) were
isolated (see "Experimental Procedures" for isolation). They were
subjected to restriction mapping, plasmid subcloning, and DNA
sequencing analysis. The relationship between the human ACAT cDNA
K1 and the four human ACAT-1 genomic DNA fragments inserted in P1
clones are shown in Fig. 3. Three of
these inserts (P1 733, 734, and 774) were found to be partially
overlapping (Fig. 3). The P1 774 insert contains the short 5'-UTR
sequence, the entire coding region, and the 3'-UTR (designated as exons 1-16) of the human ACAT-1 gene. The P1 4651 insert contains only the
long 5'-UTR (designated as exon Xa) and not the short
5'-UTR or the coding region of the human ACAT-1 gene. The sequence
CCGAATTCGG (designated as exon Xb) found in human ACAT
cDNA K1 nt 1280-1289 and the 4.3-kb ACAT-1 mRNA has not been
located within these genomic clones at present.
To test the biological activity, DNAs prepared from clone P1 774 were
co-transfected along with the plasmid DNA pcDNAneo to perform
stable transfection experiments, using the ACAT-deficient CHO cell
mutant (clone AC29) as the recipient. The resultant uncloned transfectant cells (designated as uncloned P1F) were assayed for ability to synthesize cholesteryl esters in intact cells. The value was
compared with values obtained in AC29 cells and in 25RA cells (the
parental CHO cells of AC29 cells) (8, 34). The results (Table I)
indicate that the P1 774 insert was able to stimulate cholesteryl ester
synthesis in AC29 cells. Control experiments indicate that the P1
vector without the insert along with pcDNAneo did not stimulate
cholesteryl ester synthesis in the AC29 cells (results not shown). The
P1F transfectant populations were selected by their growth resistance
to the drug G418 present in the medium. The drug G418 selected for
clones that maintained the pcDNAneo plasmid in their cell genomes.
However, each of these clones might lose the P1 774 insert during
continuous cell growth. We therefore cloned a number of stable
transfectant cells from the uncloned P1F cell population and assayed
each of these clones for its ability to synthesize cholesteryl esters.
The results (Table I) indicate that the
cloned P1F transfectant population could be divided into two
categories; clones P1F 1-4 showed near full complementation of ACAT
deficiency, while clones P1F 7-12 showed no complementation. We next
performed Southern analysis and found that the P1 774 insert was
present in clones P1F 1-4 but absent in clones P1F 7-12 (result not
shown). These results suggest that the P1 774 insert contains all of
the elements necessary for expressing the human ACAT-1 gene in CHO
cells.
The restriction map of the human genomic DNA insert in clone P1 774 is
shown in Fig. 4A. Sequencing
analysis shows that this insert contains the exonic sequences (exons
1-16) present in human ACAT cDNA K1 nt 1290-4011; however, it
does not contain the first 1289-nt sequence present at the long 5'-UTR
of human ACAT cDNA K1. The restriction map of the insert in clone
P1 4651 is shown in Fig. 4B. Sequencing analysis shows that
this insert contains 1277 (present as a single uninterrupted sequence,
exon Xa) of the first 1279 nt at the 5'-end of human ACAT
cDNA K1. The first two nucleotides (two guanines, found in nt 1 and
2 of human ACAT cDNA K1) were not found in the genomic DNA. This
difference may be the result of a mRNA capping event coupled with
the addition of an extra guanine at the 5'-end by reverse transcriptase
during the cDNA synthesis step. Other possibilities also exist. In
the human ACAT cDNA K1 sequence, a 10-bp sequence CCGAATTCGG (exon Xb) is present immediately downstream from the 1279 bp (the
cDNA sequence flanking the 10-bp sequence is shown in Fig. 1).
Repeated cloning and sequencing work has indicated that the exon
Xb sequence has not been localized downstream to the exon
Xa sequence in the P1 4651 insert or upstream from the exon
1 sequence in the P1 774 insert. Furthermore, the restriction enzyme
analyses showed that the 3'-end of P1 4651 insert (more than 20 kb
upstream from exon 1; Fig. 4B) and the 5'-end of P1 774 insert (Fig. 4A) do not overlap with each other.
Exon/Intron Organization--
Restriction fragments of the P1
774 insert and the P1 4651 insert were subcloned and sized. The sizes
of the introns were estimated by compiling the sizes of the subcloned
fragments; for larger fragments (greater than 4 kb), the fragments were
further subcloned and sized to confirm the size estimation. The human genomic DNA fragments containing the exonic sequences present in the
4.0-kb human ACAT cDNA K1 were sequenced to localize exons. All
exon-flanking regions were sequenced bidirectionally. Table II provides available exon-intron
boundary nucleotide sequences, exon lengths, sizes of the respective
intervening introns, and the amino acid interrupted by each intron. The
exonic sequences in the human genomic DNA were in almost complete
agreement with the published human ACAT cDNA K1 sequence (10).
Repeated sequencing of the human ACAT-1 genomic DNA revealed that two
errors were made in the previous assignment of the cDNA sequence.
The nucleotides coding amino acid 207 should be GCC coding alanine
instead of CGC coding arginine as previously published; also, the
nucleotides coding amino acid 475 should be CTC instead of CTG (the
coding amino acid at 475 remains as leucine). With the exception of
intron 3, all introns determined begin with a 5'-GT and conclude with a
3'-AG terminus. However, intron 3 was found to contain a rare 5'-GC
splice donor junction. The sequence immediately downstream from the
1277-bp exon Xa (aagtcaaaaa) and the sequence immediately upstream from the 99-bp exon 1 (tgggcgccag) do not conform to any known
intronic sequences specific for RNA splicing reactions. Other salient
features of the human ACAT genomic structure include protein coding
beginning in exon 2 and terminating in exon 16. Introns 1-3 are large,
each longer than 7 kb. The nucleotide sequences flanking exon
Xb in the genomic DNA have not been identified at present.
Identification of the P1 and P7 Promoters--
The 648- and the
612-bp genomic DNA fragments flanking exon 1 or exon Xa of
the human ACAT-1 gene have been subcloned and sequenced (Fig.
5). To test the promoter activities,
these two fragments were each ligated into the luciferase reporter gene vector pXP-1 or pGL2-E in forward orientation. The resultant plasmids were each transiently co-transfected into the CHO cell line AC29 or the
human cell line A293. The results show that both fragments can promote
the expression of the luciferase reporter gene (result not shown). The
sequences in these two fragments have been designated as the P1 and P7
promoters. The P1 promoter sequence in the first 648 bp flanking the
exon 1 is rich in GC content (Fig. 5A). Neither a typical
TATA box nor a typical CCAAT box was found. There is a potential
Sp1-binding site; this is consistent with other TATA-less promoters, in
which transcription initiation directed by these templates is
critically dependent on Sp1 (35). This region also contains a 9-bp
sequence for the B1 transcription enhancer element within the apo-E
promoter (36); a 6-bp sequence required for transcription of the
HMG-CoA reductase gene (37); and the 11-bp motif (in inverted
orientation) known to bind the transcription factor NF- Determination of Transcription Initiation Sites and the 5'-Ends
of Human ACAT-1 mRNA--
5'-RACE was performed using the 5'-SLIC
strategy (described under "Experimental Procedures"). Total RNAs
used as templates were prepared from HeLa cells, human U937 cells, or
various native human tissues as indicated. After ligation reaction with
the anchor olignucleotide, a human ACAT-1-specific primer (ACAT22,
located in human ACAT cDNA K1 nt 1359-1374) was used as the
3'-reverse primer in PCR experiments (Fig.
6A). In numerous experiments, the results consistently gave rise to two types of products: product A
of approximately 150-160 bp and product B of approximately 1400 bp
(lane 1 in Fig. 6C; lanes
1-2 in Fig. 6D). Lanes
2-5 in Fig. 6C show results of various negative
control experiments; if yeast tRNA or H2O were used to
replace human RNAs in the 5'-SLIC reactions (lanes
2-3), if HeLa cell total RNA without going through the 5'-SLIC reaction was directly subjected to PCR amplification
(lane 4), or if the ligation product using HeLa
cell total RNA was subjected to PCR amplification with an irrelevant
primer set (lane 5), no PCR product was obtained.
For product A, two cloned PCR products were analyzed by sequencing; the
longer one (157 bp) starts with a cytosine 45 bp in the genomic DNA
sequence upstream from exon 1, while the other is 5 nt shorter
(illustrated at the top of Fig. 6B; indicated by
an asterisk in Fig. 5A). For product B, one
cloned PCR product was analyzed by sequencing. The sequence at the
5'-end of cloned product B matched identically with that of the 5'-end
of human ACAT cDNA K1, including the first two guanines (nt 1 and 2 in ACAT cDNA K1; result not shown), exon Xa, exon Xb, and part of exon 1 (illustrated at the
bottom of Fig. 6B). The 10-bp sequence (exon
Xb) located immediately downstream from the exon
Xa sequence in human ACAT cDNA K1 has also been found in the cloned product B (illustrated at the bottom of Fig.
6B).
The 5'-end of the human ACAT-1 mRNA that contains exon
Xa was also determined by primer extension analysis, using
specific reverse primer located at human ACAT cDNA K1 nt 92-71 as
probe (see "Experimental Procedures"). As shown in Fig.
7, the size of the main product is 91 nt,
which is one nucleotide shorter than the 5'-sequence starting from nt
92 present in the human ACAT cDNA K1. This difference can be
accounted for by an extra guanine present at the 5'-end of the
cDNA; this nucleotide may be added by the reverse transcriptase
action during the cDNA synthesis step. The results of the primer
extension experiment essentially corroborate with the results of the
5'-RACE experiments, indicating that the 5'-end of human ACAT cDNA
K1 is full-length. The transcription start sites for the two different
promoters are indicated by an asterisk in Fig. 5,
A and B.
Mapping the Exon 1-16 Region and the Optional Exon
Xa Region of the Human ACAT-1 Gene--
Restriction enzyme
analyses have shown that the 3'-end of P1 4651 and the 5'-end of P1 774 do not overlap with each other (Fig. 4). This raises the possibility
that the chromosomal locations of these two genomic DNA segments may be
very distant from each other. To test this possibility, we performed
FISH experiments to metaphase chromosomes and found that the P1 774 insert, containing the exon 1-16 region of the human ACAT-1 gene, is
mapped to chromosome 1q 25 (results not shown), confirming our earlier
report (12). To our surprise, the P1 4651 insert, containing the
optional exon Xa region of the human ACAT-1 gene, is mapped
to chromosome 7q 31.3 (Fig. 8). To test
the fidelity of the FISH results, we next performed PCR analysis using
genomic DNAs prepared from human cells, mouse cells, or human mouse
somatic hybrids as templates. Two sets of primers were used (described
in Fig. 9, A and
B). The first set contained sequences flanking the 385 bp
within the exon Xa region (forward primer designated as
K1T7D, 5'-GGCAGTAGACTCATCT-3'; reverse primer designated as C1-7,
5'-GATAACCCACTGGAAG-3'). The second set contained sequences flanking
the 1064-bp region within the intron 1/exon 2 junction (forward primer
designated as ACAT 30, 5'-GCACTTAGTAGATACT-3'; reverse primer
designated as C1-5, 5'-CCATTACTAGGTGTCT-3'). The results showed that
when the first primer set was used (Fig. 9C,
left), a single 385-bp PCR product was obtained when genomic
DNAs from human fibroblasts (lane HF) or mouse A9
cells containing human chromosome 7 (lane A9-7) were used
as the templates; no PCR product was found when genomic DNAs from mouse
A9 cells containing human chromosome 1 (lane A9-1) or from
the mouse A9 cells (lane Mouse) were used as templates. These results were further validated by sequencing analysis, which revealed that the 385-bp PCR product shown in lane
A9-7 is identical to the sequence found in the
corresponding region in human genomic P1 4651 insert. Conversely, when
the second primer set was used (Fig. 9C, right), a single
1064-bp PCR product was obtained when genomic DNAs from human
fibroblasts (lane HF) or mouse A9 cells containing human chromosome 1 (lane A9-1) were used as
templates; no PCR product was found when genomic DNAs from mouse A9
cells containing human chromosome 7 (lane A9-7) or from the
mouse A9 cells (lane Mouse) were used as templates. Again,
these results were further validated by sequencing analysis, which
revealed that the 1064-bp PCR product shown in lane A9-1 is
identical to the sequence found in the corresponding region in the
human genomic P1 774 insert. Along with Fig. 8, these results indicate
that the exon 1-16 region of the human ACAT-1 gene is located in
chromosome 1, while the optional exon Xa region of the
human ACAT-1 gene is located in chromosome 7.
RNase Protection Assay--
To verify the existence of the two
types of human ACAT-1 transcripts from the two promoters (P1 and P7) as
well as the existence of the mRNA that contains the exon
Xa and Xb, we performed an RNase protection
assay. A uniformly labeled 365-nt antisense riboprobe was synthesized
by in vitro transcription of the cloned human ACAT-1
cDNA fragment (nt 1113-1374, encompassing 167 nt at the 3'-end of
exon Xa, the 10 nt in exon Xb, and 85 nt at the
5'-end of exon 1 (Fig. 10A).
This probe was annealed to RNAs prepared from human fetal brain, human
fetal kidney, or yeast and then digested with RNase. When human RNAs
were used, three major protected fragments of 262, 167, and 85 bp in
length were found (Fig. 10B, lanes 1 and 2). If yeast tRNA was used in hybridization, no
protected band of discrete size was detectable (Fig. 10B,
lane 3). These results indicate that three
different protected bands can be detected. Additional experiments
obtained the same three protected fragments if RNAs from adult human
tissues were analyzed by the same assay described here (results not
shown).
Attempts to Detect the Chimeric DNA Present in the Human Genome
at Low Level through DNA Rearrangement--
It remains possible that
the long 5'-UTR (exons Xa, Xb, and 1) present
in the 4.3-kb ACAT-1 mRNA is the result of transcription from a
chimeric DNA present in the human genome; this chimeric DNA might be
produced through DNA rearrangement by certain novel mechanism(s). Such
an event, if occurring only at the low frequency, would not be
detectable by the gene mapping methods employed in Figs. 8 and 9. To
test this possibility, we designed a PCR experiment that is sensitive
enough to detect the chimeric DNA. The results are described in Fig.
11; the diagram on the
right in A predicts that if the hypothetical
chimeric DNA existed in the human genome, then a distinct 1388-bp PCR
product should have been obtained if human genomic DNAs were used as
the template and appropriate primers as indicated were used. The
results show that we could not detect the 1388-bp product by using as
much as 1000 ng of human genomic DNA as the template (B,
lane 11); in contrast, the control experiments
showed that we could detect the 1388-bp PCR product if 0.002 ng of the
plasmid DNA ACAT cDNA K1, which contains contiguous sequences of
exon Xa, exon Xb, and exon 1, was used as the
template (lane 14). To further demonstrate the
sensitivity and fidelity of our PCR method, various additional control
experiments were also planned. The left and
middle diagrams of Fig. 11A illustrate the expected sizes of the PCR products in various control lanes. The
results show that under the PCR conditions employed, we could detect
the appropriate PCR products by using as little as 0.1 ng of human
genomic DNA serving as the template (lanes 1-3
and lanes 6-8). The results of additional
control experiments (shown in lanes 5 and
10) have demonstrated the fidelity of the PCR primers used
in lanes 1-3 and 6-8. Together, the
results show that the 4.3-kb ACAT-1 mRNA is not the result of
transcription from a chimeric DNA present at low level.
Dinucleotide Repeat Polymorphism in the Human ACAT-1
Gene--
As shown in Fig. 12, intron
12 of the human ACAT-1 gene contains a stretch of GT repeats 28 bp
(boldface and underlined sequences) away from the intron 12/exon 13 (underlined
sequences) junction. To examine the polymorphism of this
repeat, genomic DNAs from 372 individuals were used to perform PCR
using two primers flanking the GT repeat region (boldface
and italic sequences). The major allele,
containing 14 GT repeats, was confirmed by sequencing. We next
attempted to use this marker to correlate possible variation in ACAT-1
gene expression with occurrence in carotid artery atherosclerosis in
145 subjects (41). We studied the GT allele distribution among cases
and controls and found that the GT allele distribution among the cases,
controls, and total sampling population (372 individuals) are
remarkably similar (results not shown). The diagram (Fig.
13) shows the distribution of the GT
repeats within the total sampling population in this study.
Four ACAT-1 mRNAs of discrete sizes (7.0, 4.3, 3.6, and 2.8 kb) have been seen consistently in various human cells and tissues (10,
21, 22, 24). Northern analyses suggest that these mRNAs share the
same coding region. In addition, using poly(A)+ mRNAs
from human HepG2 cells as template and primers flanking the entire
human ACAT-1 coding region, Matsuda et al. (22) showed that
the RT-PCR analysis consistently yielded only one detectable product of
1.7 kb, representing the size of the full-length coding region. These
data strongly suggest that the multiple mRNAs produced from the
human ACAT-1 gene contain the same coding sequence. The 3.6- and 2.8-kb
mRNAs contain the exon 1 sequence followed by the coding sequence
(exons 2-15). The 4.3-kb mRNA contains exons 1-15 and an
additional long 5'-UTR 1289 nt in length, designated as exon
Xa and Xb. The 7.0-kb mRNA contains the
exon 1 sequence, the coding sequence, but it lacks the optional exon
Xa sequence. At the genomic DNA level, the genomic P1 774 insert contains the exon 1 sequence and the entire coding region of the
human ACAT-1 gene and has been found to express human ACAT-1 activity
upon transfecting into the ACAT-deficient CHO cell mutant. This insert spans approximately 84 kb; it contains the P1 promoter region, the
entire coding region, and the 3'-untranslated region. The first introns
(1-3) are all longer than 7 kb. A rarely observed exon/intron junction
sequence, beginning with a 5'-GC, is present in intron 3 (Table II).
Several genes have been reported with introns sharing this feature (for
examples, see 42-44). For vertebrates, an extensive compilation of 5'
splice junction sequences led to the formulation of a longer,
near-consensus sequence downstream from the 5'-GU dinucleotides:
5'-(A/G)AGU-3' (reviewed in Ref. 45), this sequence is identical to the
sequence downstream of the human ACAT-1 intron 3 splice donor site. The
648-bp genomic sequence immediately upstream from the exon 1 has been
subcloned, sequenced, and identified to be the P1 promoter region of
the human ACAT-1 gene. The 5'-RACE results show that with the P1
promoter, transcription starts at the two cytosines located 45 and 40 bp away from exon 1 in the genomic DNA. Since the 7.0-, 3.6-, and 2.8-kb human ACAT-1 mRNAs contain the exon 1 sequence and the coding sequence but do not contain the long 5'-UTR of human ACAT cDNA K1, we believe that they are produced by the P1 promoter. The
difference in length between the 3.6- and the 2.8-kb mRNA may be
caused by the difference(s) in their 3'-UTRs. The longest human ACAT-1
mRNA (7.0 kb) is also consistently detected as a minor species; it
comprises approximately 5-15% of total transcripts in most tissues or
cell lines examined thus far. At present, we are unable to determine
its origin. Since it lacks exon Xa, this large RNA species
may be an incompletely spliced intermediate. Other possibilities also exist.
The 4.3-kb mRNA contains the optional exon Xa (1279 bp)
and exon Xb (10 bp), followed by exon 1 as its long 5'-UTR.
For exon Xa, 1277 of the first 1279 bp in the 5'-UTR of
human ACAT cDNA K1 have been located as a single uninterrupted
sequence in the human genomic P1 4651 insert. Using this human genomic
P1 4651 insert, the 612-bp genomic sequence immediately upstream from the 1277-bp exon Xa sequence has been subcloned, sequenced,
and identified as the P7 promoter region of the human ACAT-1 gene. The
5'-RACE and the primer extension results show that with the P7
promoter, transcription starts at the guanine at the 5'-end of exon
Xa (illustrated at the bottom of Fig.
6B and in Fig. 5B). The first two nucleotides
(two guanines) of the human ACAT cDNA K1 were not found in the
genomic DNA sequence. This difference may be the result of an mRNA
capping event coupled with adding an extra guanine at the 5'-end
of cDNA by the reverse transcriptase during the cDNA synthesis
step. Other explanations cannot be ruled out at present. The 10-bp
sequence (nt 1280-1289 in human ACAT cDNA K1; designated as exon
Xb) located immediately downstream from the exon
Xa sequence in the human ACAT cDNA K1 has not been found in the cloned genomic DNAs.
Gene mapping analyses by FISH experiments and PCR analyses of
human-mouse somatic cell hybrids indicate that the exon 1 region the
coding region and the P1 promoter region of the human ACAT-1 gene is
located in chromosome 1, while the P7 promoter and the optional exon
Xa region are located in chromosome 7. These results suggest that the 4.3-kb mRNA is produced from two different
chromosomes. To test the validity of this interpretation, we performed
RNase protection assays. Three protected fragments of 85, 167, and 262 bp, have been detected, leading to the following interpretation. The
85-bp band comes from the transcript by the P1 promoter; the 167-bp
band comes from the transcript by the P7 promoter; and the 262-bp band
comes from the 4.3-kb mRNA. Together, these results show that the
4.3-kb mRNA is produced from two different chromosomes. We also
considered the alternative interpretation that the 4.3-kb mRNA may
be a transcript of a chimeric DNA present at low levels in the human
genome; the chimeric DNA would be produced by certain novel cellular
mechanism(s). However, this interpretation is not supported by the
results reported in Fig. 11.
The result of the RNase protection assay suggests that the 4.3-kb
ACAT-1 mRNA is produced by a novel RNA recombination event that
takes place between the two discontinuous RNAs transcribed by the P1
promoter and the P7 promoter. The molecular nature of the recombination
event is not clear at present. It may involve trans-splicing reactions.
Trans-splicing is a posttranscriptional event that joins two
discontinuous transcripts intermolecularly to produce a mature mRNA
species (for reviews, see Refs. 46-48). While there are ample examples
of trans-splicing that occur in lower organisms (including
trypanosomes, nematodes, and plants), to our knowledge, only a few
potential examples have been reported in mammalian species. Perbal and
colleagues (49-51) showed that in chicken and human thymic cells, the
expression of a mature c-myb protooncogene mRNA may
require the intermolecular linkage of coding sequences localized to two
different chromosomes. Sullivan et al. (52) showed the
possible existence of a mRNA in fetal rat liver resulting from
linkage of coding sequences from the androgen-binding protein gene,
localized in chromosome 10, and the histidine decarboxylase gene,
localized in chromosome 3. It should be pointed out that in these
studies, the existence of two precursor transcripts that were required
to produce the mature mRNA by trans-splicing was never demonstrated
by RNase protection assays. No further progress could be found in the
literature following these initial studies, which were published in the
early 1990s. Eul et al. (53) showed that a pre-mRNA
transcribed from a SV 40 viral DNA can form a mature mRNA species
via trans-splicing in intact mammalian cells as well as in HeLa cell
nuclear extracts in vitro. This report shows that the
biochemical machinery required to produce trans-splicing is present in
extracts of mammalian cells. These investigators used viral
pre-mRNA, not cellular pre-mRNA, as the substrate. More
recently, Caudevilla et al. (54) demonstrated that natural
trans-splicing of pre-mRNAs of a cellular gene (carnitine octanoyltransferase) does occur in vitro. The 4.3-kb ACAT-1
mRNA described in this report may be the result of trans-splicing. However, we note that there is not an obvious 5'-splice site present at
the junction of exon Xa and the RNA immediately downstream from it. Therefore, if the 4.3-kb ACAT-1 mRNA is produced through trans-splicing reactions, the detailed molecular mechanism involved must deviate from that of the normal RNA trans-splicing reactions demonstrated in the literature. The most intriguing and puzzling aspect
of our finding is that the 10-bp palindromic sequence in human ACAT-1
cDNA K1 nt 1280-1289, designated as exon Xb, has not
been found within the genomic clones. It is possible that this sequence
may play important role(s) in a novel trans-splicing reaction. Further
investigations are needed to clarify these issues. In addition, further
investigations are needed to evaluate the physiological significance of
the 4.3-kb ACAT-1 mRNA.
The finding that human ACAT-1 gene contains two different promoter
elements, with the P1 promoter located within the genomic region
immediately upstream from transcription start sites, bears resemblance
to a number of other mammalian genes reported (55-57). Our studies
using various deletion constructs (results not shown) suggested that
the two promoters may contain different cis-acting elements,
implying that the two human ACAT-1 promoters may be controlled by
different trans-acting factors under different physiological conditions. This possibility will be pursued in the future.
A GT dinucleotide polymorphism has been identified within intron 12. We
attempted to use this marker to correlate possible variation in the
human ACAT-1 gene expression with occurrence in carotid artery
atherosclerosis and found that the distribution of the GT repeats
within the cases and the controls in our sampling population is
identical. Since our sampling population is small and geographically
restricted, our results cannot be definitively generalized. In the
future, the human ACAT-1 gene polymorphic marker reported here may
prove useful for correlating variation of human ACAT-1 gene expression
with various genetic abnormalities in lipid metabolism and in human diseases.
This is the first report describing the genomic organization of ACAT
genes. The implication that the 4.3-kb human ACAT-1 mRNA is derived
from two discontinuous precursor RNAs produced from two different
chromosomes provides a novel system for further studies on RNA
recombination reactions in mammalian species. In addition, other
results described here will be useful for further studies on the
regulation of the human ACAT-1 gene and its products.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
DNA fragments were
used as the ladder. The PCR products were subcloned into pBluescript
SK+ vector or T-vector and sequenced. For each cloned PCR
product, approximately 150 bp from each end were sequenced.
-32P]CTP. The transcription reaction with
T3 RNA polymerase was performed in vitro using a
kit from Promega. Total RNAs isolated from human fetal brain or fetal
kidney, at 100 µg each, were hybridized to radiolabeled riboprobe
(2 × 105 cpm) at 42° for 14 h. The hybridized
samples were digested with 1000 units of RNase T1 at 37° for 15 min.
After ethanol precipitation, samples were analyzed by electrophoresis
followed by autoradiography.
603/+65 region
containing the P1 promoter were isolated from a subclone of P1 774. Similarly, various fragments within the
612/+150 region containing
the P7 promoter were isolated from a subclone of P1 4651. These
fragments were subcloned into the multiple cloning site of the
luciferase reporter gene vector pXP1 (29) or vector pGL2-E (Promega).
Constructs containing the promoters were identified by restriction
enzyme analysis and by sequencing. Transfection was performed by either the calcium phosphate method (30) or the LipofectAMINE method (Life
Technologies, Inc.). 5 µg of the luciferase reporter gene constructs
were co-transfected with 5 µg of the pSV-
-galactosidase plasmids
(Promega) into the CHO cell line AC29 or human cell line A293 grown to
approximately 50% confluency in six-well plates. 48 h after
transfection, cells were harvested for luciferase (31) and
-galactosidase (32) activity assays. Luciferase activities were
normalized to the
-galactosidase values to account for variations in
transfection efficiency.
). The MAXI Script kit (Ambion) was used
for labeling the riboprobes. The RNA blots for Northern analyses were
prepared according to procedures previously described (10).
Hybridization signals were analyzed using a PhosphorImager (Molecular Dynamics).
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Fig. 1.
The 429-bp sequence present within the 5'-UTR
of human ACAT-1 cDNA is present in ACAT-1 poly(A)+ RNAs
of human brain, intestine, and liver. A, analysis of
RT-PCR products, using the human ACAT cDNA K1 nt 982-997 (sequence
GCACGGGTTAAGATCCT) as the forward primer and nt 1410-1395
(sequence TCTTCACCCACCATTG) as the reverse primer. The templates used
for the reverse transcription steps were poly(A)+ RNAs
isolated from various indicated human tissues as indicated. Control
experiments showed that if poly+ RNAs from mouse liver or
yeast tRNA were used as the template, no PCR products were detectable.
The results described here are typical of five different experiments.
B, partial sequence of the RT-PCR products that include the
10-bp palindromic sequence CCGAATTCGG (human ACAT cDNA K1 nt
1280-1289).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Northern analysis of various human ACAT-1
mRNAs. Melanoma cell poly(A)+ RNAs (4 µg/lane)
was probed with each of the 32P-labeled riboprobes from
various indicated fragments of human ACAT cDNA K1. For simplicity,
only representative results are shown. Lane 1,
probed with Cod1, Cod2, Cod3, or Cod4; lane 2,
probed with 3'U1; lane 3, probed with 5'U1. Each
probe above is shown to hybridize with all four ACAT-1 mRNAs.
Lane 4, probed with 5'U2 or 5'U3; each of these
two probes hybridizes with the second band (4.3 kb). The length and the
coverage of the probes are as follows: 5'U1, 85 bp (1290-1374); 5'U2,
215 bp (982-1196); 5'U3, 534 bp (769-1302); Cod1, 781 bp
(1282-2062); Cod2, 723 bp (2062-2784); Cod3, 267 bp (2784-3050);
Cod4, 398 bp (2062-2459); 3'U1, 223 bp (3100-3322). Nucleotides are
numbered according to human ACAT cDNA K1 (10).
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Fig. 3.
The relationship between human ACAT cDNA
K1 and ACAT-1 genomic DNAs inserted into P1 733, P1 734, P1 774, and P1
4651. Exons are indicated by Arabic
numerals. The location of the 10-bp palindromic sequence
CCGAATTCGG remains unknown.
Synthesis of cholesteryl ester in uncloned and individually cloned
P1F transfectant cells
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Fig. 4.
Restriction maps of human ACAT-1 gene.
A, P1 774; B, P1 4651. Abbreviations for various
restriction enzymes are indicated. Lengths were drawn to scale. The
locations of early introns (introns 1-3) and early exons within these
inserts were indicated.
Exon-intron organization of the human ACAT-1
gene
, found
within the promoter of the TNF-
gene (38). In the P7 promoter
sequence, within the first 612-bp genomic DNA fragment flanking the
optional exon Xa (Fig. 5B), a typical TATA box
is also absent. However, two copies of a typical CCAAT box are found in
this region. Other potential binding sites for various transcription
factors include Sp1, Ap2, and TFIID (in inverted orientation) (39); the
insulin enhancer sequence is also present (40).
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Fig. 5.
Sequences of human ACAT-1 promoter
regions. A, P1 promoter; B, P7 promoter.
Potential transcriptional elements as predicted by sequence analysis
are underlined. The asterisks represent the
transcription start sites, with the upstream asterisk
designated as the +1 position. The boldface and
underlined sequences are the exonic sequences in
human ACAT cDNA K1 sequence.
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Fig. 6.
5'-RACE of human ACAT-1 mRNAs using the
5'-SLIC strategy. Procedures to carry out 5'-RACE using the
5'-SLIC strategy are described under "Experimental Procedures."
A, diagram of the overall strategy used. B,
diagram outlining the primer used in 5'-SLIC and the sizes of products
obtained from the short mRNAs (A1, A2) or
from the long mRNA (B). The sequences of 5'-RACE
products A1 and A2 are the same as that indicated in exon 1 (uppercase) and its 5'-flanking region
(lowercase) obtained by sequence analysis of human ACAT-1
genomic DNA inserted into P1 774; the sequence of 5'-RACE product B is
the same as that of 5'-UTR (uppercase) containing exons
Xa, Xb, and 1 of human ACAT cDNA K1; the
5'-flanking region (lowercase) of exon Xa was
obtained by sequence analysis of human ACAT-1 genomic DNA inserted into
P1 4651. C and D, size analyses of the 5'-RACE
products using the 5'-SLIC strategy. For C, lane
1, HeLa cell total RNA was used; lanes
2-5 were performed as negative controls; in lane
2 or 3, HeLa cell total RNA was replaced with
yeast tRNA or double distilled H2O; in lane
4, HeLa cell total RNA without the 5'-SLIC reaction was
directly subjected to PCR amplification; in lane
5, the ligation product using HeLa cell total RNA was
subjected to PCR amplification with an irrelevant primer set (ACAT30,
ACAT29), which was located within human ACAT-1 intron 1. Lane M, 1-kb DNA ladder from Life Technologies,
Inc. For D, lane 1, total RNAs
prepared from human fetal kidney were used as the templates;
lane 2, total RNAs prepared from fetal brain
tissue were used as templates. Lane M, DNA
ladders (Sino-American Biotech Co.).
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Fig. 7.
Primer extension analysis. The human
ACAT-1 specific reverse primer 5'-ATCCCAGCACTTTAGGAGGCCG-3'(human ACAT
cDNA K1 nt 92-71) was used as probe; the human liver mRNA was
used as the template. In a separate experiment, the same primer was
used to produce a sequencing ladder, using human ACAT genomic DNA
inserted into P1 4651 as the template. Both the primer extension
product and the sequence ladder were analyzed by a sequencing gel. In
this gel, the TGCA lanes on the left show the sequencing
ladder; lane 1 on the right shows the primer
extension products. The estimated sizes of the two primer extension
products are as indicated by arrows.
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Fig. 8.
In situ hybridization of
biotin-labeled human ACAT genomic DNA inserted into P1 4651 to human
metaphase chromosomes. The left panel shows
an idiogram of human chromosome 7. The right
panel shows a digital image of human ACAT genomic DNA
inserted into P1 4651 to partial metaphase chromosomes of a normal
individual. Arrows indicate the probe signal at 7 q31.3. No
signal was detected at other chromosome locations.
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Fig. 9.
Location of human ACAT-1 coding region and
ACAT-1 optional exon Xa region by PCR analyses, using
genomic DNAs from human fibroblast cells (HF), mouse
A9 cells (mouse), mouse A9 cells containing human chromosome 1 (A9-1),
or mouse A9 cells containing human chromosome 7 (A9-7) as
templates. The primers for PCR analysis are indicated in
A and B. In C, the left
gel shows results obtained using primers indicated in
A; the right gel shows results
obtained using primers indicated in B.
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Fig. 10.
Analysis of human ACAT-1 mRNAs by RNase
protection assay. A, illustration of the predicted
result. This diagram predicts that three protected fragments
of different size should be observed, if trans-splicing event occurs
(see text for details). B, the actual result of the RNase
protection assay; RNAs from different sources were used for hybridizing
with the riboprobe. Lane 1, human fetal brain
RNA; lane 2, human fetal kidney RNA;
lane 3, yeast tRNA; lane 4,
yeast tRNA (the sample mixture after hybridization was not digested
with RNase); lane 5, sequencing ladders of
unrelated DNA serving as size markers. The RNase protection assay was
conducted according to procedures described under "Experimental
Procedures." The results shown here are representative of two
separate experiments.
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Fig. 11.
Attempts to detect the
hypothetical chimeric DNA at low level by PCR. Diagrams
in A illustrate the predicted results by PCR. The actual
results are shown in B. DNAs in various quantities from
different sources as indicated were used as the template for the PCR
experiment. 1000, 100, or 0 ng of genomic DNA from HEK293 cells
(lanes 11-13) or 0.002 ng of the plasmid DNA
containing the full-length sequence human ACAT cDNA K1 (lane
14), was used as the template; the primer set, L8 (GTA GAG ACG GGG
TTT CAC CG; located in exon Xa, nt 3-22 of cDNA K1)
and DP2 (CGG CAG CGG GCA CTT CGG CCA A; located in exon 1, 1353-1374
nt of cDNA K1), was used. Lane M, PCR size
markers. 100, 10, 0.1, or 0 ng of genomic DNA of human cell HEK293
(lanes 1-4) and 0.1 ng of P1 DNA 4651 known to
contain the uninterrupted exon Xa of human ACAT-1
(lane 5) was used as the template; the primer
set, K1T7D (GGC AGT AGA CTC ATC T; located at exon Xa, nt
812-827 of cDNA K1) and C1-7 (GAT AAC CCA CTG GAA G; located at
exon Xa, nt 1181-1196 of cDNA K1), was used. 100, 10, 0.1, or 0 ng of genomic DNA of human cell HEK293 (lanes
6-9) and 0.1 ng of P1 DNA 774 known to contain the human
ACAT-1 coding region (lane 10) was used as the
template; the primer set, ACAT30 (GCA CTT AGT AGA TAC T; located at
human ACAT intron 1) and C1-5 (CCA TTA CTA GGT GTC T; located at human
ACAT exon 2, nt 1500-1515 of cDNA K1), was used. PCR experiments
were performed in a 100-µl volume containing 50 mM KCl,
10 mM Tris-HCl, pH 8.3, 1.5 mM
MgCl2, 200 µM each dNTP, 400 nM
each specific primer, and 5 units of Taq polymerase. For
lanes 1-5, the reactions were run as follows:
cycle 1, 3 min at 94 °C; cycles 2-32, 45 s at 94 °C,
40 s at 51 °C, and 50 s at 72 °C; cycle 33, 10 min at
72 °C. For lanes 6-10, the annealing
temperature was reduced to 49 °C; for lanes
11-14, the annealing temperature was raised to 61 °C;
other PCR conditions used were the same as described for
lanes 1-5. After PCR, the products were analyzed
along with size markers on 1.2% agarose gel.
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Fig. 12.
Sequence of the (GT)n
microsatellite-containing region within intron 12 of the ACAT-1
gene. The sequences of GT repeat (boldface
type) and exon 13 are underlined. The two
boldface and italic sequences are the
two primers used to analyze the polymorphism of this repeat sequence
among the human population.
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Fig. 13.
Frequency distribution of the (GT)n
alleles within the human ACAT-1 gene in a sample of 372 individuals. Allele designations correspond to the number of
dinucleotide repeats.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. Dan Schroen and Matt Vincenti for providing advice for using the pXP1 vector for ACAT promoter analysis. We also thank Nancy Nutile-McMenemy, Chris LaPointe, Eddie Wong, and Charlie Coon for participating in certain stages of this work. We acknowledge critical comments made by the reviewers of this manuscript and thank Dr. Charles Cole for stimulating discussion and helpful advice. The oligonucleotides used in this work were synthesized by Sushima Rampal at Beckman Instruments using the Beckman Oligo 1000 synthesizer and at the Shanghai Institute of Biochemistry, China.
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FOOTNOTES |
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* This work was supported by National Institutes of Health (NIH) Grants HL 36709 and HL 60306 (to T. Y. C.), National Natural Scientific Foundation of China Grant 39425005 (to B.-L. L.), and NIH Grant HL 16512 (to L. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence may be addressed: Shanghai Institute of Biochemistry, Shanghai, China. Fax: 86-21-6433-8357.
Visiting scientist from the Laboratory of Molecular Virology,
Shanghai Medical University, Shanghai, China.
§§ To whom correspondence may be addressed: Dept. of Biochemistry, Dartmouth Medical School, Hanover, NH 03755. Tel.: 603-650-1622; Fax: 603-650-1128.
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ABBREVIATIONS |
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The abbreviations used are: ACAT, acyl-CoA:cholesterol acyltransferase; FISH, fluorescence in situ hybridization; RACE, rapid amplification of the cDNA ends; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; kb, kilobase(s); bp, base pair(s); UTR, untranslated region; nt, nucleotide(s); CHO, Chinese hamster ovary; SLIC, single strand ligation to single strand cDNA.
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