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Originally published In Press as doi:10.1074/jbc.M006322200 on July 28, 2000
J. Biol. Chem., Vol. 275, Issue 41, 31739-31746, October 13, 2000
Novel Expression of Equivocal Messages Containing Both Regions of
Choline/Ethanolamine Kinase and Muscle Type Carnitine
Palmitoyltransferase I*
Naoshi
Yamazaki,
Yasuo
Shinohara,
Kazuaki
Kajimoto,
Masayuki
Shindo, and
Hiroshi
Terada
From the Faculty of Pharmaceutical Sciences, University of
Tokushima, Shomachi-1, Tokushima 770-8505, Japan
Received for publication, July 17, 2000
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ABSTRACT |
For characterization of the detailed gene
structure of human muscle type carnitine palmitoyltransferase I
(M-CPTI), we analyzed the 5'-upstream region of the M-CPTI transcripts.
As a result, we found a cDNA clone containing a nucleotide sequence
unexpected from the reported M-CPTI gene structure in the upstream
region of its 5' end. Comparison of this nucleotide sequence with that of genomic DNA showed that this sequence was derived from the 3'-untranslated region of the gene encoding choline/ethanolamine kinase- (CK/EK-) located upstream of the M-CPTI gene. Southern blot analysis showed that there was no other region homologous to the
CK/EK- gene in the whole human genome. Thus, the overlapping transcript was concluded to be produced from the functional genes of
CK/EK- and M-CPTI. Furthermore, cDNAs containing both exons of
these genes were detected by the polymerase chain reaction using the
cDNA of human heart M-CPTI obtained by specific reverse transcription from its 3'-untranslated region as a template. From these
results, the production and organization of these overlapping transcripts are discussed.
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INTRODUCTION |
For fatty acid -oxidation in mitochondria, long chain fatty
acids are transported into the mitochondrial matrix space across the
inner membrane in forms of acyl-CoAs. However, as the inner membrane is
not permeable to long chain fatty acyl-CoAs, they enter the
mitochondrial matrix space via a "carnitine system" consisting of
three steps (1, 2): 1) formation of acylcarnitines from long chain
fatty acyl-CoAs catalyzed by carnitine palmitoyltransferase I
(CPTI)1 located in the
mitochondrial outer membrane, 2) import of acylcarnitines in exchange
with carnitine in the matrix space mediated by carnitine-acylcarnitine carrier located in the inner membrane, and 3) formation of long chain
fatty acyl-CoAs from the imported acylcarnitines catalyzed by carnitine
palmitoyltransferase II located on the inner side of the inner membrane.
Because CPTI is responsible for the first rate-limiting step in
oxidation of fatty acids in mitochondria, considerable attention has
been paid to its structural and functional features (for recent review,
see Ref. 3). Two isoforms of CPTI, liver type and muscle type
(M-CPTI), are expressed in mammals. Although the primary structures of
these CPTI isoforms are similar, their Km values for
carnitine and susceptibilities to malonyl-CoA inhibition are different.
Furthermore, their tissue distributions are quite different (3). M-CPTI
is dominantly expressed in major energy consuming cells, such as
heart, skeletal muscle, brown adipose tissue, and testis. In contrast,
liver type CPTI is expressed in cells that do not show extensive energy
metabolism, such as liver, kidney, lung, intestine, and pancreas.
We first isolated cDNA clones of M-CPTI from rat brown adipose
tissue and human heart (4, 5). Later, Wang et al. (6) determined the gene structure of rat M-CPTI, in which transcription and
translation are initiated at exon 1 and exon 2, respectively. In
contrast, we (7) and van der Leij et al. (8) reported that,
in the human M-CPTI gene, there are two additional noncoding exons 1A
and 1B, which are used alternatively, in the 5'-upstream region of exon
2 containing the translation initiation codon. We suggested that the 5'
end of exon 1A and/or its upstream region as well as the 5' end of exon
1B function are transcription initiation sites (7). Subsequently, Yu
et al. (9) found that transcription is initiated
alternatively at two noncoding exons U and M located upstream of exon 2 in the human M-CPTI gene. Exon M corresponds exactly to exon 1B,
whereas the 5' end of exon U is located about 40 base pairs
downstream of the 5' end of exon 1A. These results suggested that the
transcription initiation mechanism(s) of M-CPTI is not simple.
In addition, a gene encoding a choline kinase-like protein was found to
be located only about 300 base pairs upstream of exon 1A of the human
M-CPTI gene with the same strand direction (7). Quite recently, the
gene of a mouse choline kinase like protein was reported to encode
choline/ethanolamine kinase- (CK/EK-) (10). Therefore, the gene
encoding the human choline kinase-like protein should be the human
CK/EK- gene. Because this gene is located in close proximity to the
M-CPTI gene, it is possible that there is a regulatory element(s) that
controls transcription of M-CPTI in the CK/EK- gene. For
understanding the transcription mechanism of the human M-CPTI gene, we
studied its gene structure and found novel transcripts containing exons
of both the CK/EK- and M-CPTI gene.
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EXPERIMENTAL PROCEDURES |
Materials and General Methods--
Human poly(A)+
RNAs, genomic DNA and a placental genomic DNA library were obtained
from CLONTECH (Palo Alto, CA), and T4 RNA ligase
and EX Taq polymerase were from TaKaRa Shuzo (Tokyo, Japan). All other reagents and enzymes were obtained as described previously (7). Gene-specific oligonucleotides were prepared and used as primers.
The nucleotide sequences and loci of these oligonucleotides are shown
in Table I. Recombinant experiments were carried out essentially
according to standard methods (11) or as recommend by the supplier.
Reverse Transcription of Human poly(A)+
RNAs--
Poly(A)+ RNAs of human tissues were reverse
transcribed with random hexamer, oligo(dT) primer (T17 Adp
(7)) or the human M-CPTI-specific primers. The nucleotide sequences of
human M-CPTI-specific primers P1, P2, and P3 were based on that of the
human M-CPTI gene (7). After reverse transcription, the reaction
mixtures were treated with NaOH to hydrolyze mRNA. The
single-stranded cDNAs (ss-cDNAs) obtained were used as
templates in the polymerase chain reaction (PCR), single strand
ligation to ss-cDNA-PCR (SLIC-PCR) and 3' rapid amplification of
cDNA ends (3' RACE).
Isolation of cDNA Clones of Human M-CPTI Containing Its
5'-Upstream Region by SLIC-PCR--
The cDNA clones corresponding
to the 5'-upstream region of mRNA of human M-CPTI were isolated by
SLIC-PCR (12, 13) with synthetic oligonucleotides of the adapter primer
A (3'-H2N-CCGTTACAGCTGGAGGGATGTTGGGCTTAAGGATGp-5'), adapter primer B (5'-GGCAATGTCGACCTCCCTACAAC-3'), and adapter primer C
(5'-CTCCCTACAACCCGAATTCCTAC-3'). By using T4 RNA ligase, adapter
primer A was attached to the 3' end of ss-cDNAs of human heart and
skeletal muscle obtained by reverse transcription with random hexamer
or T17 Adp. For amplifying cDNAs encoding M-CPTI, the
first PCR was carried out using adapter primer B/P4. For selecting M-CPTI cDNAs, the first PCR mixtures were diluted and used for the
second PCR, using adapter primer C/P5. SLIC-PCR products were subcloned
into plasmid vectors, and their nucleotide sequences were determined by
the chain termination method.
Reverse Transcription-PCR of the Transcript Containing CK/EK-
Gene and M-CPTI Gene--
Two kinds of ss-cDNAs of human tissues
were used as templates of PCR: 1) a mixture of reverse transcription
products obtained with random hexamer and T17 Adp and 2)
ss-cDNAs of M-CPTI obtained with the specific primers P1, P2, and
P3. To amplify cDNA corresponding to the transcript containing the
CK/EK- gene and M-CPTI gene, oligonucleotide primers of P6, P7, P8,
and P9 corresponding to various exons of the CK/EK- gene were used
in combination with primer P10 corresponding to exon 3 of the M-CPTI
gene. The primers P11 and P12 corresponding to exon 1A/U and 1B/M of
M-CPTI gene, respectively, were also used. For confirmation of the
RT-PCR products using ss-cDNA obtained by reverse transcription
with primer P3, Southern blot analysis was carried out using a cDNA
probe corresponding to exon 2 to exon 3 of human M-CPTI (positions  1
to +261 according to Ref. 5). The probe DNA was prepared by
multipriming with [ -32P]dCTP.
Southern Blot Analysis of the Human Genome--
Human genomic
DNA (10 µg) was digested with restriction enzymes, and reaction
mixtures were subjected to agarose gel electrophoresis. Then the
separated DNA fragments were transferred to nitrocellulose membranes
and hybridized with the probe. Human genomic DNA clone HG11 containing
parts of the CK/EK- gene (5, 7) was digested with BamHI
and HindIII and used as a probe after radiolabeling by the
multipriming method.
Determination of the Gene Structure of Human CK/EK---
The
genomic DNA clone HG21 containing the human CK/EK- gene was isolated
from a human placental genomic DNA library by screening with the DNA
probe used for Southern blot analysis of the human genome. The clone
HG21 was digested with appropriate restriction enzymes, and DNA
fragments were subcloned into plasmid vectors and sequenced. Three
types of PCR were employed to isolate and characterize the cDNA
encoding human CK/EK-: 1) RT-PCR using ss-cDNA of human heart
with a primer pair of P13/P14 prepared by comparison of the nucleotide
sequence of genomic DNA of human CK/EK- with those of cDNAs of
rat and mouse CK/EK- (14, 15); 2) for isolation of cDNA in the
3'-downstream region of P14, 3' RACE using ss-cDNA obtained by
reverse transcription with T17 Adp with a primer pair of
P8/Adp (7), and a subsequent second reaction using the diluted first
PCR mixture with a primer pair of P15/Adp; and 3) for isolation of
cDNA in the 5'-upstream region of P13, SLIC-PCR with primer pairs
of the adapter primers B/P16 and C/P17. cDNAs obtained by RT-PCR,
3' RACE, and SLIC-PCR were subcloned into plasmid vectors and
sequenced. From the nucleotide sequences of cDNA and genomic DNA,
the intron-exon junctions of the human CK/EK- gene were determined.
The nucleotide sequences of genomic DNA and cDNA of human CK/EK-
have been submitted to the DDBJ Nucleotide Sequence Data Base
under accession numbers AB029885 and AB029886, respectively.
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RESULTS |
SLIC-PCR Analysis of the 5'-Upstream Region of the M-CPTI
Message--
The structure of the upstream region of the human M-CPTI
gene reported to date (7, 9) is schematically summarized in Fig.
1A. In the upstream of exon 2 containing the translation initiation codon, there are two additional
exons, which are used alternatively, i.e. exon 1A and 1B
determined from the results of 5' RACE by us (7) or exon U and M from
the results of primer extension by Yu et al. (9). Exon M
corresponds to exon 1B, whereas the nucleotide of the 5' end of exon U
is the cytosine base shown by the symbol (#) located inside exon 1A. We
tried to determine the exact 5' end of the human M-CPTI message for understanding its transcription mechanism.
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Fig. 1.
Organization of the 3' region of the
CK/EK- gene and the 5' region of the M-CPTI
gene (A) and the nucleotide sequence of the clone
HHR-A10 obtained by SLIC-PCR (B). The genomic DNA
structures of human M-CPTI and CK/EK- are as reported (7).
Boxes represent exons, and amino acid coding and noncoding
regions are shown by closed and open boxes,
respectively. There are two noncoding exons in the M-CPTI gene, exons
1A/1B (7) and U/M (9). Exon M corresponds to exon 1B, whereas exon U
(shown by a cross-hatched box) exists inside exon 1A. The 5'
end of exon U reported as a transcription initiation site (9) is marked
by #. Parts of nucleotides of exon 11 of the CK/EK- gene and exon
1A/U of M-CPTI are shown by capital letters, and those of
the spacer region between the two genes are shown by lowercase
letters. The nucleotides observed in the 5' region of HHR-A10 are
marked by asterisks.
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First, we carried out SLIC-PCR. Amplified cDNAs derived from heart
and skeletal muscle were obtained by reverse transcription with random
hexamer and T17 Adp. These four cDNA samples showed almost the same electrophoretic patterns, and their nucleotide sequences were determined after subcloning. Comparison of the nucleotide sequences of the SLIC-PCR clones obtained with that of the
human M-CPTI gene (7) showed that most cDNA clones contained either
exon 1A or 1B to various extents, both being linked to exon 2, as we
reported previously (7). All clones containing exon 1B/M contained its
5' terminus or more downstream region, whereas clones containing exon
1A were diverse, starting at more upstream and downstream regions of
exon U. Interestingly, the clone HHR-A10 from heart mRNA was found
to contain a sequence unexpected from the human M-CPTI gene.
Characterization of Clone HHR-A10--
The determined nucleotide
sequence of the cDNA clone HHR-A10 is shown in Fig. 1B.
It contained the sequence of exon 1A from the third cytosine, and there
were nucleotides unexpected from the sequence of the upstream region of
exon 1A, as marked by asterisks in Fig. 1, in its 5' region.
Homology analysis showed that the unexpected sequence was derived from
the 3'-untranslated region of the CK/EK- gene located upstream of
human M-CPTI, and this nucleotide chain was linked to exon 1A,
conforming to the GT-AG rule of intron/exon boundary. These results
suggested that HHR-A10 was derived from the mRNA, in which the
3'-untranslated region of the CK/EK- gene is directly linked to the
third cytosine base of exon 1A of the human M-CPTI gene by splicing
(Fig. 1A).
To confirm the existence of a transcript(s) containing exons of both
the CK/EK- gene and M-CPTI gene, we carried out RT-PCR of mRNA
of human heart and skeletal muscle using the primer pairs P6/P10 and
P7/P10, as shown in Fig. 2A.
The primer P6 corresponds to the 3'-untranslated region of the
CK/EK- gene containing the nucleotide sequence of the HHR-A10 clone
marked by an asterisk, P7 corresponds to a more upstream
region, and P10 corresponds to exon 3 of the M-CPTI gene. On agarose
gel electrophoreogram of these RT-PCR products, we detected two
significant bands caused by amplified products in both heart and
skeletal muscle with both the primer pairs P6/P10 and P7/P10, as shown
in Fig. 2B.
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Fig. 2.
RT-PCR of mRNA between the regions of the
CK/EK- and M-CPTI genes of human heart and
skeletal muscle. ss-cDNAs of human heart and skeletal muscle
obtained by reverse transcription with either random hexamer or
T17 Adp were mixed and used as templates for PCR. PCR was
performed using the primer pairs P6/P10 and P7/P10. A,
Coding (closed boxes) and noncoding (open boxes)
regions of both genes. The loci of P6 and P7 in the 3'-untranslated
region of the CK/EK- gene are shown by a hatched box. The
nucleotides marked by asterisks are those found in the 5'
region of the clone HHR-A10 (see Fig. 1). B, results of
agarose gel electrophoresis of the RT-PCR products stained by ethidium
bromide. The organization of cDNAs are also shown. For details, see
text.
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Nucleotide sequence analysis showed that these products from both
sources contained the 3'-untranslated region of exon 11 of the
CK/EK- gene and exons of the M-CPTI gene, although their organizations were different. Namely, the PCR product 1 from the slower
migration band contained the 3'-untranslated region of the CK/EK-
gene and exon 1A, exon 2, and exon 3 of the M-CPTI gene without exon
1B, as in HHR-A10. Here again, the first two nucleotides of exon 1A
were deleted according to the GT-AG rule. The PCR product 2 from the
faster migration band contained the same CK/EK- gene region as that
of product 1, and its 3' end correctly linked to exon 2 and 3 of the
M-CPTI gene, conforming to the GT-AG rule. All the nucleotide sequences
of exons of the M-CPTI region in both products were completely the same
as those reported (7). These results clearly showed that clone HHR-A10 from heart was not an artifact of SLIC-PCR and that transcripts containing both exons of these two genes really existed in human heart
and skeletal muscle in considerable amounts. In addition, as the band
intensity of product 1 was greater than that of product 2 in
preparations from both sources, the splicing between exon 11 of
CK/EK- and exon 1A of M-CPTI was suggested to take place more easily
than that between exon 11 and exon 2.
Characterization of the CK/EK- Gene Located 5'-Upstream of the
M-CPTI Gene--
As described above, we found transcripts containing
exons of both the CK/EK- gene and M-CPTI gene in heart and skeletal
muscle. The gene structure of human M-CPTI has been well studied
(7-9), and its localization is reported to be on chromosome 22q13.3
(16). On the other hand, the human CK/EK- gene has not been studied extensively, but it is registered as a choline kinase isolog (384D8_3) on human chromosome 22q13 BAC clone CIT987SK-384D8 (data base accession
number U62317). Therefore, before performing detailed analysis of
overlapping transcripts, we characterized the human CK/EK- gene.
First, we performed Southern blot analysis of the CK/EK- gene. A
genomic DNA fragment of the 3' region of the human CK/EK- gene, the
organization of which was estimated from the structure of the mouse
gene (10), was prepared as a probe (Fig.
3A). This was hybridized with
samples of human genomic DNA digested with various restriction enzymes.
As shown in Fig. 3B, the probe DNA was hybridized with only
one DNA fragment in all samples. Furthermore, the lengths of all the
hybridized DNA fragments were in agreement with those expected from the
nucleotide sequence of the registered sequence in the human chromosome
22q13 BAC clone CIT987SK-384D8, as shown in Fig. 3C. The
results of Southern blot analysis showed that there is no other gene
having a similar nucleotide sequence to that of the 3' region of
CK/EK- used as a probe and that CK/EK- is encoded only by the
gene located in the upstream region of the M-CPTI gene in the human
genome. Thus, we concluded that the possible formation of overlapping
transcripts from the other genomic region was excluded and that the
overlapping transcripts were derived from functional genes of CK/EK-
and M-CPTI located on chromosome 22q13.
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Fig. 3.
Southern blot analysis of the human
CK/EK- gene. Human genomic DNA fragments
obtained by digestion with various restriction enzymes were subjected
to agarose gel electrophoresis and then transferred to nitrocellulose
membranes and hybridized with the probe, which was prepared by
digestion of the 3' region of the CK/EK- gene with BamHI
and HindIII. A, locus of the probe DNA of the
CK/EK- gene, the organization of which is shown in Fig. 2.
B, autoradiogram of Southern blot analysis. Bars
beside the autoradiogram represent the electrophoretic bands of size
makers ( /styI). C, lengths and loci of DNA fragments
hybridized with the probe estimated from the nucleotide sequence of
human chromosome 22q13 BAC clone CIT987SK-384D8. For instance, in the
case of digestion with ApaI, the probe was expected to
hybridize with a 3115-base pair DNA fragment. kbp, kilobase
pairs.
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Comparison of the registered nucleotide sequence of the 22q13 BAC clone
CIT987SK-384D8 with those of rat and mouse CK/EK- cDNAs (14, 15)
suggested that there was a misreading in the registered nucleotide
sequence. To determine the exact gene structure, we isolated the
genomic DNA and cDNA of human CK/EK-. Because the genomic DNA
clone HG11 isolated by us (5, 7) did not contain the whole region of
the CK/EK- gene, a human genomic DNA library was screened again and
HG21 clone was isolated. By comparison of the nucleotide sequence of
genomic DNA with that of cDNA obtained by RT-PCR, 3' RACE, and
SLIC-PCR, we determined the structure of the human CK/EK- gene. The
gene organization and nucleotide sequence of cDNA of human
CK/EK- determined are shown in Fig. 4
(A and B, respectively). In addition, the
detailed structures of the 3' region of the CK/EK- gene and 5'
region of M-CPTI are shown in Fig. 5.
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Fig. 4.
Structure of the human
CK/EK- gene. A, organization
of the human CK/EK- gene and its downstream regions. Exons shown by
closed, open, and hatched boxes are as
for Fig. 2. Primers P13 and P14 were used for RT-PCR to obtain partial
CK/EK- cDNA, primers P8 and P15 were used for 3' RACE to
characterize the 3' region of CK/EK- cDNA, and primers P16 and
P17 were used for SLIC-PCR to characterize the 5' region of CK/EK-
cDNA. B, nucleotide sequence of cDNA encoding human
CK/EK-. Numbers of nucleotides are shown on the left
margin, the adenine base in the translation initiation codon being
numbered +1. The translation termination codon is shown by an
asterisk. The nucleotide sequence of the
underlined AATAAA represents a poly(A) additional
signal. The deduced amino acids are shown by the one-letter
abbreviation code under the nucleotides. Arrowheads
represent intron insertion sites.
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Fig. 5.
Detailed structure of the junction region of
the human CK/EK- gene and M-CPTI gene.
The nucleotide sequence between the 3' region of the CK/EK- gene and
the 5' region of M-CPTI gene is shown. Nucleotides in exons and introns
are shown by capital and lowercase letters,
respectively, and the deduced amino acids are shown by the one-letter
abbreviation codes under the nucleotides. Nucleotides are numbered
taking the adenine base in the translation initiation codon of human
M-CPTI as +1, and their numbers are shown on the left margin
(7). Arrows represent splice sites in the overlapping
transcript. Closed, open, and hatched
boxes are as for Fig. 2. In exon 11 of the CK/EK- gene, the
translation termination codon is marked by an asterisk, the
nucleotide sequence of the underlined AATAAA represents a
poly(A) additional signal, and the 3' end was determined by 3' RACE.
The transcription initiation site of exon U of the M-CPTI gene (9) is
marked by #.
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The gene encoding human CK/EK- was found to consist of at least 11 exons and was suggested to encode a protein of about 45 kDa consisting
of 395 amino acids, like those of rat and mouse, which encode a protein
of 45 kDa having 394 amino acids (14, 15). Furthermore, the amino acid
sequence of human CK/EK- was about 86% homologous with those of rat
and mouse. A comparison of the nucleotide sequence determined by us
with that of the 22q13 BAC clone showed that one guanine base at
position +43 in exon 1 (here the nucleotide sequence was numbered
taking the adenine base in the putative translation initiation codon as
+1) was skipped in the BAC clone. Because of this misreading, the human
CK/EK- gene (choline kinase isolog) in the registered data has been
predicted to encode a 49.7-kDa protein having 433 amino acids.
Characterization of the Overlapping Transcripts--
In the first
part of this study, we used a mixture of reverse transcription products
obtained with random hexamer and T17 Adp as a template and
amplified cDNA fragments containing both regions of the human
CK/EK- gene and M-CPTI gene (Fig. 2). To characterize the
organization of the overlapping transcripts, RT-PCR with several primer
pairs seemed to be efficient. However, the cDNA corresponding to
the overlapping transcripts might also be artificially formed by PCR,
when a particular region of the genomic DNA is transcribed into two
different messages and reverse transcription is carried out with random
primer or oligo(dT) primer, as shown in Fig.
6. To determine the exact organization of
these overlapping transcripts, PCR should be performed using a template prepared with several specific primers.
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Fig. 6.
Possible artificial amplification of cDNA
between overlapping genes X and Y. The organizations of
overlapping genes are shown at the top, with the
hatched box representing the overlapping region. Short
horizontal arrows represent the loci and directions of primers
used for PCR. A, both genes are transcribed in the same
strand direction, and both transcripts contain the nucleotides in the
hatched region. B, both transcripts are reverse transcribed
using a gene nonspecific primer, such as random primer and oligo(dT)
primer. In the figure, the reverse transcription with a random primer
is shown. Broken arrows represent the first strand
cDNAs. C, second strand cDNA of gene X is formed
using a downstream primer. D, the 3' region of the second
strand cDNA of gene X anneals with the 3' region of the first
strand cDNA of gene Y. E and F,
double-stranded cDNA that could be a template for artificial
amplification is produced. G, cDNA corresponding to a
transcript that does not exist is formed. Therefore, the first strand
cDNA should be prepared with gene-specific primer to characterize
the organization of overlapping transcripts.
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Therefore, we prepared ss-cDNAs of human M-CPTI by reverse
transcription with oligonucleotides P1, P2, and P3, which correspond to
exons 4, 15, and 20 of M-CPTI, respectively (Table
I), and performed PCR with these
ss-cDNAs as specific templates. Namely, the messages expressed in
human heart were reverse transcribed with the primer P1, P2, or P3, all
of which specifically annealed the message of M-CPTI, and PCRs were
performed using these ss-cDNAs as templates with various primer
pairs shown in Fig. 7A and
Table I. The results of agarose gel electrophoresis of PCR products stained with ethidium bromide are shown in Fig. 7B. When
ss-cDNAs obtained by reverse transcription of mRNAs with
primers P1 and P2, referred to as P1 and P2 ss-cDNAs, respectively,
were used as templates, only a significant band of the expected size
from the gene structure of M-CPTI was observed in each amplification by
primer pairs of P11/P10 and P12/P10. Nucleotide sequence analyses of
these products showed that they contained exons 1A, 2, and 3 of the
M-CPT1 gene and exons 1B, 2, and 3, respectively. In contrast, two
bands, a significant band with lower electrophoretic mobility and a
weak band with higher mobility, were detected with the primer pairs
P7/P10, P8/P10, and P9/P10. After subcloning, sequencing of the
products giving the strong band with P9/P10 using P1 and P2
ss-cDNAs showed that both products contained exons 9, 10, and 11 of
the CK/EK- gene and exons 1A, 2, and 3 of the M-CPTI gene. In
addition, both products giving the weak band contained the same exons
of both genes, but exon 1A was deleted. In these products, the 3' end
of the CK/EK- region was connected by proper splicing to the 5' end
of the M-CPTI region in similar manners to those shown in Fig.
2B. From the electrophoretic mobilities, the products of
P7/P10 and P8/P10 with P1 and P2 ss-cDNAs were expected to have
similar overlapping structures.
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Fig. 7.
Results of RT-PCR of the overlapping region
of the CK/EK- and M-CPTI genes.
A, RT-PCR specific to M-CPTI messages was performed
with the primers shown by arrowheads with reference to the
CK/EK- and M-CPTI genes, in which boxes represent exons
according to Fig. 2. B, PCR products were subjected to
agarose gel electrophoresis, and gels were stained with ethidium
bromide. C, RT-PCR products using P3 ss-cDNA were
transferred to a nitrocellulose membrane, and Southern blot analysis
was performed by hybridization of the products with the probe.
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When ss-cDNA prepared by reverse transcription with P3 (P3
ss-cDNA) was used as a template, a product derived from the M-CPTI message was detected with both the primer pairs P11/P10 and P12/P10 as
in RT-PCR with P1 and P2 ss-cDNA, but no clear electrophoretic bands were observed with primer pairs of P7/P10, P8/P10, and P9/P10 (Fig. 7B). However, as shown in Fig. 7C, two
products having expected sizes in all PCRs with P3 ss-cDNA were
detected by Southern blot analysis. The nucleotide sequences of the
products of P9/P10 with P3 ss-cDNA showed that they contained the
same sequences as those of corresponding products of P1 and P2
ss-cDNAs. Because primer P3 corresponds to exon 20 of the M-CPTI
gene, the products of P9/P10 with P3 ss-cDNA should be derived from
messages containing the sequence between exon 9 of the CK/EK- gene
and exon 20 of the M-CPTI gene, although their amounts were low (Fig.
7A). These messages were the most possible extended
transcripts covering the entire region of the RT-PCR performed in this study.
The amounts of PCR products obtained with the primer pairs P11/P10 and
P12/P10 using P1, P2, and P3 ss-cDNAs were always greater than
those obtained with the primer pairs P7/P10, P8/P10, and P9/P10 (Fig.
7B). Therefore, the structural gene of human M-CPTI starts
mainly at either exon 1A/U or 1B/M, as reported (7, 9). However, PCR
products were distinctly observed with the primer pair P9/P10 with P1,
P2, and P3 ss-cDNAs, showing that there were definite amounts of
overlapping transcripts of the CK/EK- gene and M-CPTI gene. In
addition, the band intensities of RT-PCR products with P2 ss-cDNA
were lower than those with P1 ss-cDNA, and those with P3
ss-cDNA were significantly lower than those with P1 and P2
ss-cDNAs. This could be due, at least in part, to the fact that
reverse transcription became inefficient as its range became wider.
| |
DISCUSSION |
Previously, we isolated a cDNA clone containing exon 1A of the
human M-CPTI gene by 5' RACE (7). Subsequently, Yu et al. (9) reported that transcription starts at the 5' end of exon U located
inside exon 1A. To determine the exact gene structure of human M-CPTI,
in this study we isolated various cDNA clones containing the
5'-upstream region of M-CPTI by SLIC-PCR instead of 5' RACE.
Unexpectedly, we isolated a clone HHR-A10 from heart mRNA contained
the 3'-untranslated region of the CK/EK- gene. RT-PCR (Fig. 2) and
Southern blot analysis of the human genome (Fig. 3) showed that there
were distinct transcripts containing both exons of the functional
CK/EK- and M-CPTI genes in human heart and skeletal muscle. In the
overlapping transcripts containing exon 1A of M-CPTI, the first two
nucleotides adenine and guanine of exon 1A were always excluded because
of splicing according to the GT-AG rule. Therefore, it is possible that
the reported cDNA, which started at the 5' end of exon 1A obtained
by 5' RACE (7), was not derived from overlapping transcripts and could have been derived from another transcript initiated at exon 1A or a
more upstream region. These results suggested that the transcription of
M-CPTI is "loosely" controlled and is initiated at multiple sites.
It should be noted that because of the existence of definite amounts of
the overlapping transcripts, the amount of mRNA of human M-CPTI
will be estimated as higher than it really is, when the amount is
determined from a part of its message by such methods as RT-PCR and
RNase protection assay.
Overlapping genes are commonly observed in prokaryotes, bacteriophages,
and viruses (17). However, they are quite rare in mammals. In 1986, Williams and Fried (18) first found the overlapping of two unknown
mouse genes with opposite directions. In this case, both strands of a
particular genomic region of 133 bases were utilized as templates for
transcription. Overlapping transcription using the same DNA strand in
mammals, like those of the human CK/EK- and M-CPTI genes, was first
reported for genes of the bovine myosin I heavy chain-like protein and
preprotachykinin B by Hoshimaru and Nakanishi (19). Subsequently,
overlapping genes with the same strand direction were reported for the
mouse RNA polymerase II promoter motif and the first intron of the
-glucuronidase gene (20) and the vesicular acetylcholine transporter
gene and choline acetyltransferase gene in humans and rats (21, 22). Of
these, the genes of the myosin I heavy chain-like protein and preprotachykinin B are located close together, and the overlapping region is transcribed in both messages. However, unlike the overlapping transcripts of CK/EK- and M-CPTI genes, the splice sites in this overlapping region are not completely the same, resulting in the formation of exons with different nucleotide sequences in both genes.
There should be at least four possibilities for formation of such
overlapping transcripts between CK/EK- and M-CPTI genes, as shown in
Fig. 8. Namely, 1) incomplete termination
of the CK/EK- message, in which transcription does not terminate at
the 3' end of CK/EK- gene but proceeds to certain regions of the
M-CPTI gene such as to a cluster of poly(A) additional signals in the intron between exon 7 and exon 8 (7) (possibility A); 2) transcription of the M-CPTI gene from certain regions of CK/EK- through the 3' end
of the M-CPTI gene (possibility B); 3) transcription initiated at
certain regions of CK/EK- gene and termination in a certain region
of the M-CPTI gene such as at its intron between exons 7 and 8 (possibility C); and 4) transcription initiated from the transcription
initiation site of the CK/EK- gene to the 3' end of the M-CPTI gene
(possibility D). In the case of possibility D, a message containing
both the open reading frames of CK/EK- and M-CPTI should be
produced.
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|
Fig. 8.
Possible manners of overlapping transcript
production between the CK/EK- gene and M-CPTI
gene. Ordinarily, messages consisting solely of CK/EK- and
M-CPTI are transcribed. Overlapping transcripts will be formed by
transcription from the transcription initiation site of CK/EK- to
certain regions of the M-CPTI gene (A), from certain regions
of the CK/EK- gene to the 3' end of the M-CPTI gene (B),
from certain regions of the CK/EK- gene to certain regions of M-CPTI
(C), and from the transcription initiation site of the CK/EK- gene
to the 3' end of the M-CPTI gene (D).
|
|
The results of PCRs in various regions between exon 9 of the CK/EK-
gene and exon 3 of the M-CPTI gene using specific ss-cDNAs of M-CPT
as templates showed that most messages of human M-CPTI started at exon
1A/U and exon 1B/M. In addition, we obtained cDNAs containing the
region between exon 9 of the CK/EK- gene and exon 3 of the M-CPTI
gene in PCR using P3 ss-cDNA (Fig. 7). Because primer P3
corresponds to exon 20 located downstream of the translation termination codon in exon 19 of the M-CPTI gene, we concluded that
there are overlapping transcripts containing the region between exon 9 of the CK/EK- gene and exon 20 of the M-CPTI gene, which is the most
possible extended transcript of RT-PCR in this study. These overlapping
transcripts contain the open reading frame of M-CPTI (possibilities B
and D). The amounts of RT-PCR products with P3 ss-cDNA were
apparently much lower than those with P1 and P2 ss-cDNAs
corresponding to exon 4 and exon 15 of the M-CPTI gene, respectively.
This could be due to either or both the facts that: 1) there is a
3'-splice variant lacking exon 20 of the M-CPTI gene (8) that cannot be
used as a template of reverse transcription with primer P3 and 2) the
production of ss-cDNA from the 3' region of the M-CPTI transcript
using P3 primer is more difficult than those using P1 and P2 primers
because of a wide range of reverse transcription of more than 2 kilobases of its entire message. In addition to these overlapping
transcripts, there could be overlapping transcripts that terminated
between primers P1 and P2 (possibilities A and C). However, this seems
unlikely from the fact that the intensities of electrophoretic bands of
the products using P2 ss-cDNA were not significantly lower than
those using P1 ss-cDNA. The lower amounts of the products with P2
ss-cDNA could be a result of inefficient reverse transcription
covering a wider range from exon 15.
Because the nucleotide chains of overlapping transcripts between exon 9 of the CK/EK- gene and exon 3 of the M-CPTI gene were spliced as
observed with "ordinary messages" of both genes with the exception
of their boundary region, it is possible that the message transcribed
from the transcription initiation site of the CK/EK- gene proceeds
to the region of the M-CPTI gene. Accordingly, there could be an
overlapping transcript containing the region from the 5' end of the
CK/EK- gene to the 3' end of the M-CPTI gene (possibility D).
Then how are these overlapping transcripts produced? The 3' end of the
human CK/EK- gene is located about 300 and 1000 base pairs upstream
of the 5' ends of exon 1A and exon 2 of the M-CPTI gene, respectively.
With such a proximate organization of the two genes, the transcription
of the CK/EK- gene may proceed to the M-CPTI gene, and splicing may
take place between the last exon 11 of CK/EK- and exon 1A or exon 2 of M-CPTI before addition of the poly(A) tail or without termination of
transcription. Therefore, the human CK/EK- gene and the proximate
M-CPTI gene should be useful for understanding the mechanisms of
transcription termination and splicing of a transcribed message.
The messages of the M-CPTI region were concluded to be heterogeneous
having multiple transcription initiation sites and loose transcription
termination of the CK/EK- gene. Studies on the control mechanism of
gene expression of M-CPTI as well as the physiological roles of these
overlapping transcripts are underway.
| |
FOOTNOTES |
*
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB029885 and AB029886.
To whom correspondence should be addressed. Fax: 81-88-633-9511;
E-mail: hterada@ph.tokushima-u.ac.jp.
Published, JBC Papers in Press, July 28, 2000, DOI 10.1074/jbc.M006322200
| |
ABBREVIATIONS |
The abbreviations used are:
CPTI, carnitine
palmitoyltransferase I;
M-CPTI, muscle type CPTI;
CK/EK-, choline/ethanolamine kinase-;
PCR, polymerase chain reaction;
RACE, rapid amplification of cDNA ends;
RT, reverse transcription;
SLIC-PCR, single strand ligation to ss-cDNA-PCR;
ss-cDNA, single-stranded cDNA.
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ABSTRACT
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