Proc. Natd. Acad. Sci. USA Vol. 89, pp. 12122-12126, December 1992 Biochemistry
Genomic structure of the human caldesmon gene (dfetatn/smooh m e/acmyos/tr
yosl/c d )
KEN'ICHIRO HAYASHI*, HAJIME YANO*, TAKASHI HASHIDAt, RIE TAKEUCHIt, OSAMU TAKEDAt, Kiyozo ASADAt, EI-ICHI TAKAHASHI*, IKUNOSHIN KATOt, AND KENJI SOBUE*§ *Department of Neurochemistry and Neuropharmacology, Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan; tBiotechnology Research Laboratories, Takara Shuzo Company, Ltd., 341 Seta, Otsu-shi, Shiga 520-21, Japan; and *Division of Genetics, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263, Japan
Communicated by Christian Anfinsen, September 17, 1992
ABSTRACT The high molecular weight cldemon (hCaD) is predominantly expressed in smooth muscles, whereas the low molecular weight caldesmon (I-CaD) is widely distributed in nonmusce tissues and cells. The changes in CaD isoform expression are closely correlated with the phenotypic modulation of smooth muce cells. During a search for isdorm diversity of human CaDs, I-CaD cDNAs were cloned from HeLa S3 cells. HeLa i-CaD I is composed of 558 amino acids, whereas 26 amino acids (residues 202-227 for HeLa i-CaD I) i are deleted in HeLa i-CaD H. The short amino sequence of HeLa i-CaDs is different from that of fibroblast (WI-38) I-Cal) H and human aorta h-CaD. We have also identiied WI-38 I-CaD I, which contains a 26-amino acid insertion relative to WI-38 I-CaD H. To reveal the mo r events of the expressional regulation of the CaD iorms, the genomic sructure of the human CaD gene was detemiu. The human CaD gene is composed of 14 exons and was m ed to is a single locus, 7q33-q34. The 26-amino acid in cay spliced in the mRNAs for encoded in exon 4 and Is both h-CaD and i-CaDs I. Exon 3 is the exon that encodes the central repeating domain specific to h-CaD (residues 208-436) together with the common domain in all CaDs (residues 73-207 for h-CaD and WI-38 i-CaDs, and residues 68-201 for HeLa i-CaDs). The regulation of h- and i-CaD exp is thought to depend on selection of the two 5' splice sites within exon 3. Thus, the change In essio between I-CaD and h-CaD might be caused by this splicing pathway.
Caldesmon (CaD), a calmodulin- and actin-binding protein, plays a vital role in the regulation of smooth muscle and nonmuscle contraction (1, 2). Two CaD isoforms have been identified; h-CaD (high Mr, 120,000-150,000) and i-CaD (low Mr, 70,000-80,000) as judged by NaDodSO4/polyacrylamide gel electrophoresis (3-6). Sequencing studies on chicken CaD cDNAs have demonstrated that the deduced molecular weights of h- and i-CaD are in the range of 87,000-89,000 and 59,000-60,000, respectively, and that the major parts of both CaDs have identical amino acid sequences except for the insertion of the central repeating domain of the h-CaD molecule (7-10). Structural and functional analyses have revealed that the calmodulin-, actin-, and tropomyosinbinding sites contained in a region involved in the regulation of actin-myosin interaction reside within the common carboxyl-terminal domain of both CaD isoforms (9, 11). The tissue and cell distributions of the two isoforms are distinctively different, however. h-CaD is primarily found in smooth muscles, whereas i-CaD is widely distributed in nonmuscle tissues and cells. Notably, the changes in expression of the two CaD isoforms are closely correlated with phenotypic modulation of smooth muscle cells, in which h-CaD is
predominantly expressed in differentiated smooth muscle cells and is replaced by I-CaD during dedifferentiation (1214). To investigate the regulation of CaD isoform expression, we have searched for isoform diversity of human CaDs and have determined the genomic structure¶ and the chromosomal location of the CaD gene. Our studies have revealed two splice sites within exon 3 of the CaD gene. We discuss this feature in relation to the regulation of CaD isoform expression.
MATERIALS AND METHODS Cloning and Sequencing of cDNA. An oligo(dT)-primed cDNA library from HeLa S3 mRNA was screened with 32P-labeled restriction fragments originating from embryonic chicken brain I-CaD cDNA. Four positive clones carrying I-CaD cDNAs were obtained and their sequences were determined. Southern Blot Analysis. Genomic DNA (5 pg) from HeLa S3 cells or human peripheral lymphocytes was digested with restriction enzymes and the digests were electrophoresed in 0.7% agarose gels. The separated DNA fragments were blotted to nylon membranes by the method of Southern (15). The hybridization conditions with 32P-labeled HeLa i-CaD I or II cDNA fragments have been described (9). Reverse Trauscriptio-PR. The first-strand cDNA from each cell type was synthesized by using (dT)1218 and/or the antisense primer specific to the 3' noncoding sequence of human h- and I-CaD cDNAs. Primers used in this experiment were as follows: sense primer Pn, d(ATGCTGGGTGGATCCGGATC), specific to the short amino-terminal sequence of HeLa I-CaDs; antisense primer Pm, d(GTTTAAGTTTGTGGGTCATGAATTCTCC), complementary to the common sequence in all CaD isoforms, nucleotide positions 832-859 in WI-38 i-CaD II cDNA; sense primer Pn2, d(CACCATGGATGATTTTGAGCG), nucleotide positions 108128 in WI-38 i-CaD II cDNA (16); and antisense primer Pi, d(GAAGGTAGGCTTGTCTTCTTGGAGCTTTTC), complementary to the insertion sequence of the HeLa i-CaD I sense strand (Fig. 1). DNA fragments amplified by PCR (17) were separated in 1.5% agarose gels. Cha t a of Human CaD Gene. A human placental genomic library in EMBL3 was screened by hybridization with 32P-labeled probes from the HeLa I-CaD I cDNA. Restriction mapping revealed four overlapping clones (EMBL 11, SA, 111, and C4) and a nonoverlapping clone (EMBL 2) (see Fig. 3A). Restriction fragments from each Abbreviations: CaD, caldesmon; h-CaD, high molecular weight CaD; I-CaD, low molecular weight CaD. ITo whom reprint requests should be addressed. IThe nucleotide sequences reported in this paper have been deposited in the GenBank/EMBL/DDJB data base (accession nos. D90452 and D90453).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
12122
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Proc. Natd. Acad. Sci. USA 89 (1992)
clone were subcloned and sequenced. Clones carrying the exon that encodes, the amino-terminal domain of WI-38 i-CaDs or aorta h-CaD were obtained by using the cassette primer method. This method is based on the modification and improvement of the specific-primer PCR method (18, 19). The specific DNA fragment containing the target exon thus obtained was used for isolation of the genomic clone (EMBL F5). Chromosomal Mapping of Human CaD Gene. The human CaD gene was localized by using the genomic clones EMBL 2, 11, 111, and C4 as probes in a mapping system combining fluorescence in situ hybridization with R-banding (20, 21).
Pri Prn
sense
731
752__
Pil Pi
Pn2 Pm
_~af
)
Pn2
Pi
691 -670
FIG. 2. Characterization of I-CaD isoforms expressed in HeLa S3 and WI-38 cells. The reverse transcription-PCR method was done with the indicated primers sets, using first-strand cDNA from HeLa S3 and WI-38 as templates. Sizes ofthe amplified fragments are given in base pairs.
RESULTS Isoform Diversity of Human CaDs. Sequence analysis in the present study revealed the two different molecules of i-CaD (i-CaDs I and II) that originated from HeLa S3 cells. The primary structures of HeLa I-CaDs I and II in comparison with those of W138 i-CaD II (16) and human aorta h-CaD (22) are shown schematically in Fig. 1. HeLa i-CaD I and II are composed of 558 amino acids (Mr 64,252) and 532 amino acids (Mr of 61,210), respectively; 26 amino acids (residues 202227) of HeLa i-CaD I have been deleted in HeLa i-CaD II. The short amino-terminal sequences (residues 1-18) of HeLa i-CaDs are different from those of WI-38 i-CaD II and aorta h-CaD (residues 1-24). The 26-amino acid insertion in HeLa i-CaD I is found in aorta h-CaD, but not in WI38 i-CaD II. The central repeating domain specific to h-CaD (residues 208436) is deleted in all i-CaDs. To search for isoform diversity of human CaD, the reverse transcription-PCR method was introduced (Fig. 2). The primers used in this experiment are indicated in Fig. 1. The two kinds of DNA fragments [731 and 809 base pairs (bp)] were amplified from HeLa S3 mRNA by
using a HeLa-type sense primer (Pn) and the common antisense primer (Pm). The similarly amplified DNA fragments (752 and 830 bp) were obtained from WI-38 mRNA by using a WI-38-type sense primer (Pn2). The result suggests that the two i-CaD isoforms are expressed in HeLa S3 and WI-38 cells. In both cases, large and small DNA fragments would be derived from the mRNAs for the respective i-CaD with the insertion of 26 amino acids (i-CaD I) and without it (i-CaD II). Large DNA fragments were not well amplified, however. Immunoblotting of HeLa S3 and WI-38 cells revealed that the expression of I-CaD I was very low compared with that of I-CaD II (data not shown). Therefore, such amplifications would be reflected in the amount of each mRNA for I-CaD I or II. The PCR with an antisense primer specific to the HeLa I-CaD I insertion sequence (Pi) could clearly amplify a single fragment- 670- and 691-bp fragments
HeLa I-CaD 558 amino acids
-
MLGGSGSHGRRSLAALSQ 1
0Y
C5~
antisense
12123
Pm
GEEKGTKVQAKREKLQEDKPTFKKEE
18
-
202
Pi
227
HeLa I-CaD 11 532 amino acids
MLGGSGSHGRRSLAALSQ
18
1
WI-38 I-CaD 11 538 amino acids
.M
MDDFERRRELRRQKREEMRLEAER 1
24
-
Pn2 WI-38 I-CaD 564 amino acids
MDDFERRRELRRQKREEMRLEAER 1
aorta h-CaD 793 amino acids
_
MDDFERRRELRRQKREEMRLEAER 1
GEEKGTKVQAKREKLQEDKPTFKKEE
24 208
24
233
_
GEEKGTKVQAKREKLQEDKPTFKKEE 462 437
FIG. 1. Isoform diversity of human CaDs. The identical sequences in all CaD isoforms and the central repeating domain specific to aorta h-CaD are indicated by solid bars and an open bar, respectively. The short amino-terminal sequences of each isoform and the insertion sequences specific to I-CaDs I and h-CaD are shown by one-letter amino acid symbols below the bars. Primers used in PCR analysis are indicated by arrows. Numbers indicate the positions of amino acids in each CaD molecule.
12124
Biochemistry: Hayashi et al.
Proc. Nad. Acad. Sci. USA 89 (1992)
from HeLa S3 and WI-38 mRNAs, respectively. From these results, we have identified WI-38 i-CaD I with the insertion sequence in WI-38 cells (Fig. 1). The primary structures of the human CaD isoforms identified are summarized in Fig. 1. Structure of Human CaD Gene. Five positive clones were isolated from a human placental genomic library. Each clone was subjected to further characterization, and the exoncontaining fragments derived from the respective clones were subcloned and sequenced. Fig. 3A shows the genomic construction of human CaD. Four overlapping clones (EMBL 11, 5A, 111, and C4) carried most of the exons. EMBL 2 and EMBL F5 were independent clones carrying an exon encoding the short amino-terminal sequence specific to HeLa i-CaDs (residues 6-17) or WI-38 I-CaDs and aorta h-CaD (residues 1-24). Since EMBL 2 and EMBL F5 did not overlap with EMBL 11, we could not clarify the spatial relationship between exon 1 and 1'. All intron/exon junctions (Table 1) are compatible with the splice consensus sequence except for exon 3 (23). Exon 3 is constituted as follows (Fig. 4). The common domain of the CaD isoforms (residues 68-201 for HeLa i-CaDs and 73-207 for WI-38 I-CaDs and aorta h-CaD) is encoded in exon 3a, whereas the central repeating domain specific to h-CaD (residues 208-436 for aorta h-CaD) resides in exon 3b. The consensus sequence for the 5' splice site is found in the border between exon 3a and 3b (underlined in Fig. 4). Exon 4 encodes the insertion sequence specific to the two i-CaDs I and aorta h-CaD. Based on the present findings, alternative splicing pathways are summarized in Fig. 3B. Exons 2 and 5-13 are spliced in all of the mRNAs for h- and i-CaDs, and exon 4 is spliced in the mRNA for I-CaDs I and
A
FAMB[L.
EMB. F;.-.
I
h-CaD. Exon 3a is spliced in the mRNAs for all i-CaDs, whereas exon 3ab is specifically spliced in the mRNA for h-CaD. Exons 1 and 1' encode the short amino terminus specific to HeLa i-CaDs and to WI-38 I-CaDs or aorta h-CaD, respectively. Chromosomal Locus of Human CaD Gene. Southern blot analysis of genomic DNA from HeLa S3 cells and human peripheral lymphocytes with HeLa I-CaD I cDNA fragments as probes revealed identical hybridizing patterns (Fig. 5). The same result was obtained with HeLa i-CaD II cDNA fiagments as probes (data not shown). These results suggest that the CaD isoforms are encoded by a single gene. To confirm this suggestion, the chromosomal locus of the CaD gene was determined. We examined 100 (pro)metaphase plates showing a typical R-band for all clones. The efficiency of hybridization was similar among the four kinds of probe (EMBL 2, 11, 111, and C4, indicated in Fig. 3A), and the locations of the signals were the same. For example, 51% of such R-banded chromosomes exhibited complete double-spot staining with EMBL 11 as a probe. The signals were localized in band q33-q34 of the long arm of chromosome 7. No signals were detected in the other chromosomes. Thus, the CaD gene could be assigned to band 7q33-q34 (Fig. 6).
DISCUSSION In our previous study (9), we investigated the structural and functional relationships between chicken h- and I-CaDs, in which the major parts of the amino and carboxyl termini of both isoforms are completely identical sequences. The carboxyl terminus of both chicken CaDs conserve two se-
L-MBL
,Akr..a.jj
:.1-I'. 1
j
1
ii
ii
1i
L.. :~ ~ L...
..1.i
H-,
r- .U, . 1
t.1
II
.1
..IVI.. 1.1...
..i. I.
o..1 -ll .......
;J
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D. fi _
±ortasrriootrl k-iirTaar r
k1 f
t
ci
L-
i1........1
,
1.
FIG. 3. The intron/exon organization of the human CaD gene (A) and its alternative splicing pathways (B). (A) Four overlapping genomic clones and two independent clones are shown at the top. Boxes and lines indicate the exons and introns, respectively. Sizes of introns (below) are given in kilobase pairs (kbp). The introns and exon that we have not confirmed by cloning of genomic DNA are indicated by dashed lines and box, respectively. (B) The five alternative mRNA splicing pathways used to generate HeLa I-CaDs I and H, WI-38 I-CaDs I and II, and aortic smooth muscle h-CaD are shown schematically. Filled boxes represent the common exons in all CaD isoforms, and the exons encoding the short amino-terminal sequences of HeLa I-CaDs and of WI-38 i-CaDs or aorta h-CaD are indicated by shaded and hatched boxes, respectively. Open boxes represent the exons specific to h-CaD and/or I-CaDs I.
Biochemistry: Hayashi et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
Table 1. Exon organization of human CaD gene Size, 3' splice site Exon bp 5' splice site Not determined 1 >37 CGCTCTCCCAgtgagt
A L tttcagGTCCAGACAT noncoding
1'
112
ttgoagAATCGCCTAC
2
147
3
1090
R
I
A
Y
quences showing high homology with the two tropomyosinbinding regions (T1 and T2) in the troponin T molecule. We have further identified the minimum regulatory domains, which are involved in the Ca2+-dependent regulation of the actin-myosin interaction, in the carboxyl terminus of chicken h- and i-CaDs. We compared the primary structures of human
r
AAGCAGAAAGgtaagg E A E23 CCCAGAACAGgtactg A Q N72
CaDs with those of chicken CaDs. The overall sequence
identity between the two species of CaD is 65-68%. However, all CaD isoforms in different species strongly conserve the minimum regulatory domain (89% identity). In addition to this, the amino termini of all CaD isoforms (for example, residues 21-47 and 85-121 for HeLa I-CaD I and the corresponding residues of other chicken and human CaDs) also retain completely identical sequences. Therefore, these conserved domains might be important for the structure and
Q gtacagTGTGCCTGAC S V P D aaaaagGGAGAAGAGA G E E ggttagATCAAAGAEG I K D ccacagCCGCCCTGGA S R D G tcttagGAAGAGAAGA E E K tcotagATAGAAGAGC I E E ttttagCAGTGGTGTC S S G V tttcagGGAACAAAAA G T K ttgtagGAAACTGCTG E
T L
78
5
146
6
264
7
141
8
44
9
82
10
138
11
96
12
81
13
>533
R
tggoagGTTTGAGACG
AAAGAAACAGgtacag L K K Q436 AAAAGAAGAGgtaaat K K E E42 ATACTTTCAGgtaaga N T F510 CAGAGAGGAGgtaagg L R E E598 ATCTCTCAAGgtattt S S L K65 TGCAGAAAAGgtaaat V Q K659 TGCAATTGAGgtgaga S A I E687 ACCAAATAAGgtgagc T P N K733 CAAACCTTCTgtaagt P K P S765 CCCCACTAAGgtaatc S P T K792
A
tatcagGACTTGAGAC D
4
function of CaDs. To begin to elucidate the molecular events of the regulation of CaD isoform expression during phenotypic modulation of smooth muscle cells, we have investigated the genomic structure ofthe human CaD gene. The CaD gene is composed of at least 14 exons (Fig. 3) and was mapped to a single locus on 7q33-q34 by using the four kinds of probes that can cover the overall CaD gene (Fig. 6). The isoform diversity of this protein (Fig. 1) can be explained by selection of exon 1 or 1', exon 3a or 3ab, and/or exon 4. Exon 1 or 1' encodes the short amino-terminal sequences of all human CaD isoforms. Exon 4 is spliced in i-CaDs I and h-CaD. Exon 3a is also spliced in all i-CaDs, whereas exon 3ab is specifically spliced in h-CaD. Among these splicing pathways, the most interesting is the regulatory mechanism to select the two consensus sequences for the 5' splice sites in exon 3. The same exon structure as for the human CaD gene has been also found in the genome of chicken CaD (unpublished work). Such use of competing splice sites has been reported in viral transcription units of adenovirus EJA (24), simian virus 40 tumor antigen genes (25), Drosophila Ultrabithorax (26) and transformer genes (27), and the human kininogene gene (28). Among them, the two former examples have been the object of the most study
Not determined
V
Exon sequences are shown in uppercase letters, and intron sequences in lowercase letters. Amino acids encoded by each exon are indicated by one-letter symbols below the nucleotides, and their position numbers at each 5' splice site are from the human aorta h-CaD sequence (22). The sequences and position number with underline are from HeLa I-CaD sequence. I
ttatacao T GTG CCT GAC GAG GAG GCC AAG ACA ACC ACC ACA AAC ACTI CAA E E A V K T T P D T T T N Q
r-----
AG CGC CTG GCT CGG CGT E R L A R R
3a
GAG E
G AT GIG UGAA WUG G AIAT I GAG
E
V
D
G
E
D
A
CGCA TTC CG A F L
GAA AGA CGC CAA AAA CGC CTT CAG GAG GCT CTG GAG CGG CAG AAG GAG TTC GAC CCA E R R Q K R L 0 E A L E R 0 K E F D P
TA ACA GAT GCA AGT CTG TCG CTC CCA AGC AGA AGA ATG CAA AAT GAC ACA GCA GAA AAT GAA ACT ACC GAG AAG GAA T A D S L S L P S R R M 0 N D T A E N E T T E K E AA AGT GAA AGT CGC CAA GAA AGA TAC GAG ATA GAG S E S R E R E K E Q Y
GAA E
284 91
(520)
ACA 365
(601)
AT
3ab
GA" CAI!51A 0 1 V E
GTG
V
ATG m
118
(124)
GAA
446 145
(682) (151)
E
ACA GAA ACA GTC ACC AAG TCC TAC CAG AAG AAT GAT TGG AG T E T V T K S Y Q K N D W R
GTG GAA GAG AAA ACA ACT GAA AGC CAG GAG GAA
V
E
E
K
T
T
E
S
Q
E
E
V
V
M
S
L
K
N
G
Q
ATC AGT TCA GAA GAG CCT AAA CAA GAG GAG GAG AGG GAA CAA GGT TCA GAT GAG ATT TCC CAT CAT GAA AAG ATG GAA GAG S S E E P K Q E E E R E Q G S D E S H E M E H K E GAA GAC AAG GAA AGA GCT GAG GCA GAG AGG GCA AGG TTG GAA GCA GAA GAA AGA GAA AGA ATT AAA GCC GAG CAA GAC AAA E E E A E D K R A R A R E L A E E R E R 0 D K K A E AAG ATA GCA GAT GAA CGA GCA AGA ATT GAA E K A E R A R D
G00 A
GAA GAA AAA GCA GCT GCC CAA GAA AGA GAA AGG AGA GAG GCA GAA GAG E E A K A E R E A R R E E A Q E
AGG GAA AGG ATG AGG GAG GAA GAG AAA AGG GCA SCA GAG E E R E R R E K R A E A M
GAG E
AGG CAG AGG ATA AAG GAG GAA GAG AAA AGG GCA GCA GAG
0
R
R
527 (763) 1T2 (178)
GAh 608 E 1 99 ACA GTG GTA ATG TCA TTA AAA AAT GGG CAG T
K
E
E
E
K
R
A
A
(97)
T
AT GCT GAA GAA AAC AAG AAA GAA GAC AAG GAA AAG GAG GAG GAG GAA GAG GAG AAG CCA AAG CGA GGG AGC ATT GGA A E E N K K E K E K E E E D D E E E K P K R G S G N
12125
E
(844) (205) (925) (232)
(1006)
(259) (1087) (286)
(1 168) (313)
(1249) ( 340)
GAG AGG CAG AGG ATA AAG GAG GAA GAG AAA AGG GCA GCA GAG GAG AGG CAG AGG ATA AAA GAG GAA GAG AAA AGG GCA GCA 0 R E E E E Q R R K K R A E E A R K E E E R A K A
(1330) (367)
GAO E
(1411) (394)
GAA AAG GTA GAA CAG AAA ATA GAA GGG AAA TGG GTA AAT GAA AAG E K V E 0 K G E K W V N E K
(1492) (421)
GAG GAG AGG CAA AGG GCC AGG GCA GAG GAG GAA GAG AAG GCT AAG GTA GAA GAG CAG AAA CGT AAC AAG CAG CTA GAA 0 R A R A E E E E K A K V E E 0 K R N K 0 L E E E R AAA AAA CGT GCC ATG CAA GAG ACA AAG ATA AAA K K R 0 E T K K A M
000 G
AAA GCA CAA GAA GAT AAA CTT CAG ACA GCT GTC CTA AAG AAA 0 E D K L 0 T A V L K K K A
CAaYntacLata Q I
(1537)
(436)
FIG. 4. Nucleotide sequence of exon 3 (uppercase) with flanking intron sequences (lowercase). Exons 3aand 3ab are boxed. The consensus sequence of 5' splice sites for exons 3a and 3ab are underlined, and the intron/exonjunctions are indicated by arrowheads. The nucleotides and the deduced amino acid sequences from HeLa I-CaD cDNAs are numbered at right, and the numbers in parentheses are from human aorta h-CaD cDNA (22).
12126
Proc. Nad. Acad. Sci. USA 89 (1992)
Biochemistry: Hayashi et a!. BarnH n b
94 4
a b
ib
of CaD isoform expression must be studied at both the transcriptional and the mRNA processing level.
01
We thank Dr. T. Hon (National Institute of Radiological Sciences) for his suggestions. This study was partly supported by grants from the Scientific Research Fund of the Ministry of Education, Science, and Culture of Japan and from the Nissan Foundation.
A,
23.1
66
Hind ill
EcoR
~-
4~
Itd
23 2.0
FIG. 5. Southern blot analysis. Genomic DNA from HeLa S3 (lanes a) and human peripheral lymphocytes (lanes b) was digested with the indicated enzymes. Probes were made from the full-length HeLa I-CaD I cDNA. Size markers are indicated in kilobases.
according to which trans-acting factors might be involved in the modification of the recognition process of alternative 5' splice sites by U1 small nuclear ribonucleoprotein (29, 30). A splicing factor derived from HeLa nuclear extract (SF2; ref. 31) plays a critical role in selection of the 5' splice site; it promotes use of the 5' splice site that is located near the 3' splice site in an artificial mRNA precursor containing two 5' splice sites (32). An anti-SF2 factor to suppress the activation of the 5' splice site by SF2 has been also reported (33). Here we propose the selective usage of competing splice sites in relation to cell differentiation. Regulation of h- and 1-CaD expression may depend on unknown trans-acting factors which are linked to phenotypic modulation of smooth muscle cells. Further studies are required for identification of such factors. Additionally, it is necessary to determine whether HeLa- and WI-38-type mRNAs are transcribed from the same promoter or an independent promoter. The regulation
FIG. 6. Chromosome mapping of human CaD gene. A whole R-banded (pro)metaphase plate was hybridized with the biotinylated CaD gene. Arrows indicate the signals on 7q33-q34.
1. Sobue, K., Muramoto, Y., Fujita, M. & Kakiuchi, S. (1981) Proc. Natl. Acad. Sci. USA 78, 5652-5655. 2. Sobue, K" & Sellers, J. R. (1991)J. Biol. Chem. 266, 12115-12118. 3. Owada, M., Hakura, A., lida, K., Yahara, I., Sobue, K. & Kakiuchi, S. (1984) Proc. Natl. Acad. Sci. USA 81, 31333137. 4. Sobue, K., Tanaka, T., Kanda, K., Ashino, N. & Kakiuchi, S. (1985) Proc. Natl. Acad. Sci. USA 82, 5025-5029. 5. Bretcher, A. & Lynch, W. (1985) J. Cell Riol. 100, 1748-1757. 6. Dingus, J., How, S. & Bryan, J. (1986) J. Cell Biol. 102, 1748-1757. 7. Hayashi, K., Kanda, K., Kimizuka, F., Kato, I. & Sobue, K. (1989) Biochem. Biophys. Res. Commun. 164, 503-511. 8. Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R. G. & Lin, W.-G. (1989) J. Biol. Chem. 264, 13873-13879. 9. Hayashi, K., Fujio, Y., Kato, I. & Sobue, K. (1991) J. Biol. Chem. 266, 355-361. 10. Bryan, J. & Bryan, L. (1991) J. Muscle Cell Motil. 12, 372-375. 11. Wang, C.-L. A., Wang, L.-W. C., Xu, S., Lu, R. C., SaavedraAlanis, V. & Bryan, J. (1991) J. Biol. Chem. 266, 9166-9172. 12. Ueki, N., Sobue, K., Kanda, K., Hada, T. & Higashino, K. (1987) Proc. Natl. Acad. Sci. USA 84, 9049-9053. 13. Sobue, K., Kanda, K., Tanaka, T. & Ueki, N. (1988) J. Cell. Biochem. 37, 317-325. 14. Glukhova, M. A., Kabakov, A. E., Frid, M. G., Ornatsky, 0. I., Belkin, A. M., Mukhin, D. N., Orekhov, A. N., Koteliansky, V. E. & Smirnov, V. N. (1988) Proc. Natl. Acad. Sci. USA 85, 9542-9546. 15. Southern, E. (1975) J. Mol. Biol. 98, 503-517. 16. Novey, R. E., Lin, J. L.-C. & Lin, J. J.-C. (1991) J. Biol. Chem. 266, 16917-16924. 17. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K., Horn, G. T., Erlich, H. A. & Arnheim, N. (1985) Science 230, 1350-1354. 18. Kalman, M., Kalman, E. T. & Cashel, M. (1990) Biochem. Biophys. Res. Commun. 167, 504-506. 19. Shyamala, V. & Ames, G. F.-L. (1989) Gene 84, 1-8. 20. Takahashi, E., Hon, T., O'Connell, P., Leppert, M. & White, R. (1990) Hum. Genet. 86, 14-16. 21. Takahashi, E., Yamauchi, M., Tsuji, H., Hitomi, A., Meuth, M. & Hon, T. (1991) Hum. Genet. 88, 119-121. 22. Humphrey, M. B., Herrera-Sosa, H., Gonzalez, G., Lee, R. & Bryan, J. (1992) Gene 112, 197-204. 23. Mount, S. M. (1982) Nucleic Acids Res. 10, 459-472. 24. Berk, A. J. & Sharp, P. A. (1978) Cell 14, 659-711. 25. Ziff, E. B. (1982) Nature (London) 287, 491-499. 26. Beachy, P. A., Helfand, S. L. & Hogness, D. S. (1985) Nature (London) 313, 545-551. 27. Boggs, R. T., Greagor, P., Idriss, S., Belote, J. M. & McKeown, M. (1987) Cell 50, 739-747. 28. Kitamura, N., Kitagawa, H., Fukushima, D., Takag, Y., Miyata, T. & Nakanishi, S. (1985) J. Biol. Chem. 260, 8610-8617. 29. Lerner, M. R., Boyle, J. A., Mount, S. M., Wolin, S. & Steitz, J. (1980) Nature (London) 283, 220-224. 30. Rogers, J. & Wall, R. (1980) Proc. Natl. Acad. Sci. USA 77, 1877-1879. 31. Krainer, A. R., Conway, G. C. & Kozak, D. (1990) Genes Dev. 4, 1158-1171. 32. Krainer, A. R., Conway, G. C. & Kozak, D. (1990) Cell 62, 35-42. 33. Mayeda, A. & Krainer, A. R. (1992) Cell 68, 365-375.
Genomic structure of the human caldesmon gene (dfetatn/smooh m e/acmyos/tr
yosl/c d )
KEN'ICHIRO HAYASHI*, HAJIME YANO*, TAKASHI HASHIDAt, RIE TAKEUCHIt, OSAMU TAKEDAt, Kiyozo ASADAt, EI-ICHI TAKAHASHI*, IKUNOSHIN KATOt, AND KENJI SOBUE*§ *Department of Neurochemistry and Neuropharmacology, Biomedical Research Center, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan; tBiotechnology Research Laboratories, Takara Shuzo Company, Ltd., 341 Seta, Otsu-shi, Shiga 520-21, Japan; and *Division of Genetics, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263, Japan
Communicated by Christian Anfinsen, September 17, 1992
ABSTRACT The high molecular weight cldemon (hCaD) is predominantly expressed in smooth muscles, whereas the low molecular weight caldesmon (I-CaD) is widely distributed in nonmusce tissues and cells. The changes in CaD isoform expression are closely correlated with the phenotypic modulation of smooth muce cells. During a search for isdorm diversity of human CaDs, I-CaD cDNAs were cloned from HeLa S3 cells. HeLa i-CaD I is composed of 558 amino acids, whereas 26 amino acids (residues 202-227 for HeLa i-CaD I) i are deleted in HeLa i-CaD H. The short amino sequence of HeLa i-CaDs is different from that of fibroblast (WI-38) I-Cal) H and human aorta h-CaD. We have also identiied WI-38 I-CaD I, which contains a 26-amino acid insertion relative to WI-38 I-CaD H. To reveal the mo r events of the expressional regulation of the CaD iorms, the genomic sructure of the human CaD gene was detemiu. The human CaD gene is composed of 14 exons and was m ed to is a single locus, 7q33-q34. The 26-amino acid in cay spliced in the mRNAs for encoded in exon 4 and Is both h-CaD and i-CaDs I. Exon 3 is the exon that encodes the central repeating domain specific to h-CaD (residues 208-436) together with the common domain in all CaDs (residues 73-207 for h-CaD and WI-38 i-CaDs, and residues 68-201 for HeLa i-CaDs). The regulation of h- and i-CaD exp is thought to depend on selection of the two 5' splice sites within exon 3. Thus, the change In essio between I-CaD and h-CaD might be caused by this splicing pathway.
Caldesmon (CaD), a calmodulin- and actin-binding protein, plays a vital role in the regulation of smooth muscle and nonmuscle contraction (1, 2). Two CaD isoforms have been identified; h-CaD (high Mr, 120,000-150,000) and i-CaD (low Mr, 70,000-80,000) as judged by NaDodSO4/polyacrylamide gel electrophoresis (3-6). Sequencing studies on chicken CaD cDNAs have demonstrated that the deduced molecular weights of h- and i-CaD are in the range of 87,000-89,000 and 59,000-60,000, respectively, and that the major parts of both CaDs have identical amino acid sequences except for the insertion of the central repeating domain of the h-CaD molecule (7-10). Structural and functional analyses have revealed that the calmodulin-, actin-, and tropomyosinbinding sites contained in a region involved in the regulation of actin-myosin interaction reside within the common carboxyl-terminal domain of both CaD isoforms (9, 11). The tissue and cell distributions of the two isoforms are distinctively different, however. h-CaD is primarily found in smooth muscles, whereas i-CaD is widely distributed in nonmuscle tissues and cells. Notably, the changes in expression of the two CaD isoforms are closely correlated with phenotypic modulation of smooth muscle cells, in which h-CaD is
predominantly expressed in differentiated smooth muscle cells and is replaced by I-CaD during dedifferentiation (1214). To investigate the regulation of CaD isoform expression, we have searched for isoform diversity of human CaDs and have determined the genomic structure¶ and the chromosomal location of the CaD gene. Our studies have revealed two splice sites within exon 3 of the CaD gene. We discuss this feature in relation to the regulation of CaD isoform expression.
MATERIALS AND METHODS Cloning and Sequencing of cDNA. An oligo(dT)-primed cDNA library from HeLa S3 mRNA was screened with 32P-labeled restriction fragments originating from embryonic chicken brain I-CaD cDNA. Four positive clones carrying I-CaD cDNAs were obtained and their sequences were determined. Southern Blot Analysis. Genomic DNA (5 pg) from HeLa S3 cells or human peripheral lymphocytes was digested with restriction enzymes and the digests were electrophoresed in 0.7% agarose gels. The separated DNA fragments were blotted to nylon membranes by the method of Southern (15). The hybridization conditions with 32P-labeled HeLa i-CaD I or II cDNA fragments have been described (9). Reverse Trauscriptio-PR. The first-strand cDNA from each cell type was synthesized by using (dT)1218 and/or the antisense primer specific to the 3' noncoding sequence of human h- and I-CaD cDNAs. Primers used in this experiment were as follows: sense primer Pn, d(ATGCTGGGTGGATCCGGATC), specific to the short amino-terminal sequence of HeLa I-CaDs; antisense primer Pm, d(GTTTAAGTTTGTGGGTCATGAATTCTCC), complementary to the common sequence in all CaD isoforms, nucleotide positions 832-859 in WI-38 i-CaD II cDNA; sense primer Pn2, d(CACCATGGATGATTTTGAGCG), nucleotide positions 108128 in WI-38 i-CaD II cDNA (16); and antisense primer Pi, d(GAAGGTAGGCTTGTCTTCTTGGAGCTTTTC), complementary to the insertion sequence of the HeLa i-CaD I sense strand (Fig. 1). DNA fragments amplified by PCR (17) were separated in 1.5% agarose gels. Cha t a of Human CaD Gene. A human placental genomic library in EMBL3 was screened by hybridization with 32P-labeled probes from the HeLa I-CaD I cDNA. Restriction mapping revealed four overlapping clones (EMBL 11, SA, 111, and C4) and a nonoverlapping clone (EMBL 2) (see Fig. 3A). Restriction fragments from each Abbreviations: CaD, caldesmon; h-CaD, high molecular weight CaD; I-CaD, low molecular weight CaD. ITo whom reprint requests should be addressed. IThe nucleotide sequences reported in this paper have been deposited in the GenBank/EMBL/DDJB data base (accession nos. D90452 and D90453).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
12122
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Proc. Natd. Acad. Sci. USA 89 (1992)
clone were subcloned and sequenced. Clones carrying the exon that encodes, the amino-terminal domain of WI-38 i-CaDs or aorta h-CaD were obtained by using the cassette primer method. This method is based on the modification and improvement of the specific-primer PCR method (18, 19). The specific DNA fragment containing the target exon thus obtained was used for isolation of the genomic clone (EMBL F5). Chromosomal Mapping of Human CaD Gene. The human CaD gene was localized by using the genomic clones EMBL 2, 11, 111, and C4 as probes in a mapping system combining fluorescence in situ hybridization with R-banding (20, 21).
Pri Prn
sense
731
752__
Pil Pi
Pn2 Pm
_~af
)
Pn2
Pi
691 -670
FIG. 2. Characterization of I-CaD isoforms expressed in HeLa S3 and WI-38 cells. The reverse transcription-PCR method was done with the indicated primers sets, using first-strand cDNA from HeLa S3 and WI-38 as templates. Sizes ofthe amplified fragments are given in base pairs.
RESULTS Isoform Diversity of Human CaDs. Sequence analysis in the present study revealed the two different molecules of i-CaD (i-CaDs I and II) that originated from HeLa S3 cells. The primary structures of HeLa I-CaDs I and II in comparison with those of W138 i-CaD II (16) and human aorta h-CaD (22) are shown schematically in Fig. 1. HeLa i-CaD I and II are composed of 558 amino acids (Mr 64,252) and 532 amino acids (Mr of 61,210), respectively; 26 amino acids (residues 202227) of HeLa i-CaD I have been deleted in HeLa i-CaD II. The short amino-terminal sequences (residues 1-18) of HeLa i-CaDs are different from those of WI-38 i-CaD II and aorta h-CaD (residues 1-24). The 26-amino acid insertion in HeLa i-CaD I is found in aorta h-CaD, but not in WI38 i-CaD II. The central repeating domain specific to h-CaD (residues 208436) is deleted in all i-CaDs. To search for isoform diversity of human CaD, the reverse transcription-PCR method was introduced (Fig. 2). The primers used in this experiment are indicated in Fig. 1. The two kinds of DNA fragments [731 and 809 base pairs (bp)] were amplified from HeLa S3 mRNA by
using a HeLa-type sense primer (Pn) and the common antisense primer (Pm). The similarly amplified DNA fragments (752 and 830 bp) were obtained from WI-38 mRNA by using a WI-38-type sense primer (Pn2). The result suggests that the two i-CaD isoforms are expressed in HeLa S3 and WI-38 cells. In both cases, large and small DNA fragments would be derived from the mRNAs for the respective i-CaD with the insertion of 26 amino acids (i-CaD I) and without it (i-CaD II). Large DNA fragments were not well amplified, however. Immunoblotting of HeLa S3 and WI-38 cells revealed that the expression of I-CaD I was very low compared with that of I-CaD II (data not shown). Therefore, such amplifications would be reflected in the amount of each mRNA for I-CaD I or II. The PCR with an antisense primer specific to the HeLa I-CaD I insertion sequence (Pi) could clearly amplify a single fragment- 670- and 691-bp fragments
HeLa I-CaD 558 amino acids
-
MLGGSGSHGRRSLAALSQ 1
0Y
C5~
antisense
12123
Pm
GEEKGTKVQAKREKLQEDKPTFKKEE
18
-
202
Pi
227
HeLa I-CaD 11 532 amino acids
MLGGSGSHGRRSLAALSQ
18
1
WI-38 I-CaD 11 538 amino acids
.M
MDDFERRRELRRQKREEMRLEAER 1
24
-
Pn2 WI-38 I-CaD 564 amino acids
MDDFERRRELRRQKREEMRLEAER 1
aorta h-CaD 793 amino acids
_
MDDFERRRELRRQKREEMRLEAER 1
GEEKGTKVQAKREKLQEDKPTFKKEE
24 208
24
233
_
GEEKGTKVQAKREKLQEDKPTFKKEE 462 437
FIG. 1. Isoform diversity of human CaDs. The identical sequences in all CaD isoforms and the central repeating domain specific to aorta h-CaD are indicated by solid bars and an open bar, respectively. The short amino-terminal sequences of each isoform and the insertion sequences specific to I-CaDs I and h-CaD are shown by one-letter amino acid symbols below the bars. Primers used in PCR analysis are indicated by arrows. Numbers indicate the positions of amino acids in each CaD molecule.
12124
Biochemistry: Hayashi et al.
Proc. Nad. Acad. Sci. USA 89 (1992)
from HeLa S3 and WI-38 mRNAs, respectively. From these results, we have identified WI-38 i-CaD I with the insertion sequence in WI-38 cells (Fig. 1). The primary structures of the human CaD isoforms identified are summarized in Fig. 1. Structure of Human CaD Gene. Five positive clones were isolated from a human placental genomic library. Each clone was subjected to further characterization, and the exoncontaining fragments derived from the respective clones were subcloned and sequenced. Fig. 3A shows the genomic construction of human CaD. Four overlapping clones (EMBL 11, 5A, 111, and C4) carried most of the exons. EMBL 2 and EMBL F5 were independent clones carrying an exon encoding the short amino-terminal sequence specific to HeLa i-CaDs (residues 6-17) or WI-38 I-CaDs and aorta h-CaD (residues 1-24). Since EMBL 2 and EMBL F5 did not overlap with EMBL 11, we could not clarify the spatial relationship between exon 1 and 1'. All intron/exon junctions (Table 1) are compatible with the splice consensus sequence except for exon 3 (23). Exon 3 is constituted as follows (Fig. 4). The common domain of the CaD isoforms (residues 68-201 for HeLa i-CaDs and 73-207 for WI-38 I-CaDs and aorta h-CaD) is encoded in exon 3a, whereas the central repeating domain specific to h-CaD (residues 208-436 for aorta h-CaD) resides in exon 3b. The consensus sequence for the 5' splice site is found in the border between exon 3a and 3b (underlined in Fig. 4). Exon 4 encodes the insertion sequence specific to the two i-CaDs I and aorta h-CaD. Based on the present findings, alternative splicing pathways are summarized in Fig. 3B. Exons 2 and 5-13 are spliced in all of the mRNAs for h- and i-CaDs, and exon 4 is spliced in the mRNA for I-CaDs I and
A
FAMB[L.
EMB. F;.-.
I
h-CaD. Exon 3a is spliced in the mRNAs for all i-CaDs, whereas exon 3ab is specifically spliced in the mRNA for h-CaD. Exons 1 and 1' encode the short amino terminus specific to HeLa i-CaDs and to WI-38 I-CaDs or aorta h-CaD, respectively. Chromosomal Locus of Human CaD Gene. Southern blot analysis of genomic DNA from HeLa S3 cells and human peripheral lymphocytes with HeLa I-CaD I cDNA fragments as probes revealed identical hybridizing patterns (Fig. 5). The same result was obtained with HeLa i-CaD II cDNA fiagments as probes (data not shown). These results suggest that the CaD isoforms are encoded by a single gene. To confirm this suggestion, the chromosomal locus of the CaD gene was determined. We examined 100 (pro)metaphase plates showing a typical R-band for all clones. The efficiency of hybridization was similar among the four kinds of probe (EMBL 2, 11, 111, and C4, indicated in Fig. 3A), and the locations of the signals were the same. For example, 51% of such R-banded chromosomes exhibited complete double-spot staining with EMBL 11 as a probe. The signals were localized in band q33-q34 of the long arm of chromosome 7. No signals were detected in the other chromosomes. Thus, the CaD gene could be assigned to band 7q33-q34 (Fig. 6).
DISCUSSION In our previous study (9), we investigated the structural and functional relationships between chicken h- and I-CaDs, in which the major parts of the amino and carboxyl termini of both isoforms are completely identical sequences. The carboxyl terminus of both chicken CaDs conserve two se-
L-MBL
,Akr..a.jj
:.1-I'. 1
j
1
ii
ii
1i
L.. :~ ~ L...
..1.i
H-,
r- .U, . 1
t.1
II
.1
..IVI.. 1.1...
..i. I.
o..1 -ll .......
;J
..
D. fi _
±ortasrriootrl k-iirTaar r
k1 f
t
ci
L-
i1........1
,
1.
FIG. 3. The intron/exon organization of the human CaD gene (A) and its alternative splicing pathways (B). (A) Four overlapping genomic clones and two independent clones are shown at the top. Boxes and lines indicate the exons and introns, respectively. Sizes of introns (below) are given in kilobase pairs (kbp). The introns and exon that we have not confirmed by cloning of genomic DNA are indicated by dashed lines and box, respectively. (B) The five alternative mRNA splicing pathways used to generate HeLa I-CaDs I and H, WI-38 I-CaDs I and II, and aortic smooth muscle h-CaD are shown schematically. Filled boxes represent the common exons in all CaD isoforms, and the exons encoding the short amino-terminal sequences of HeLa I-CaDs and of WI-38 i-CaDs or aorta h-CaD are indicated by shaded and hatched boxes, respectively. Open boxes represent the exons specific to h-CaD and/or I-CaDs I.
Biochemistry: Hayashi et al.
Proc. Natl. Acad. Sci. USA 89 (1992)
Table 1. Exon organization of human CaD gene Size, 3' splice site Exon bp 5' splice site Not determined 1 >37 CGCTCTCCCAgtgagt
A L tttcagGTCCAGACAT noncoding
1'
112
ttgoagAATCGCCTAC
2
147
3
1090
R
I
A
Y
quences showing high homology with the two tropomyosinbinding regions (T1 and T2) in the troponin T molecule. We have further identified the minimum regulatory domains, which are involved in the Ca2+-dependent regulation of the actin-myosin interaction, in the carboxyl terminus of chicken h- and i-CaDs. We compared the primary structures of human
r
AAGCAGAAAGgtaagg E A E23 CCCAGAACAGgtactg A Q N72
CaDs with those of chicken CaDs. The overall sequence
identity between the two species of CaD is 65-68%. However, all CaD isoforms in different species strongly conserve the minimum regulatory domain (89% identity). In addition to this, the amino termini of all CaD isoforms (for example, residues 21-47 and 85-121 for HeLa I-CaD I and the corresponding residues of other chicken and human CaDs) also retain completely identical sequences. Therefore, these conserved domains might be important for the structure and
Q gtacagTGTGCCTGAC S V P D aaaaagGGAGAAGAGA G E E ggttagATCAAAGAEG I K D ccacagCCGCCCTGGA S R D G tcttagGAAGAGAAGA E E K tcotagATAGAAGAGC I E E ttttagCAGTGGTGTC S S G V tttcagGGAACAAAAA G T K ttgtagGAAACTGCTG E
T L
78
5
146
6
264
7
141
8
44
9
82
10
138
11
96
12
81
13
>533
R
tggoagGTTTGAGACG
AAAGAAACAGgtacag L K K Q436 AAAAGAAGAGgtaaat K K E E42 ATACTTTCAGgtaaga N T F510 CAGAGAGGAGgtaagg L R E E598 ATCTCTCAAGgtattt S S L K65 TGCAGAAAAGgtaaat V Q K659 TGCAATTGAGgtgaga S A I E687 ACCAAATAAGgtgagc T P N K733 CAAACCTTCTgtaagt P K P S765 CCCCACTAAGgtaatc S P T K792
A
tatcagGACTTGAGAC D
4
function of CaDs. To begin to elucidate the molecular events of the regulation of CaD isoform expression during phenotypic modulation of smooth muscle cells, we have investigated the genomic structure ofthe human CaD gene. The CaD gene is composed of at least 14 exons (Fig. 3) and was mapped to a single locus on 7q33-q34 by using the four kinds of probes that can cover the overall CaD gene (Fig. 6). The isoform diversity of this protein (Fig. 1) can be explained by selection of exon 1 or 1', exon 3a or 3ab, and/or exon 4. Exon 1 or 1' encodes the short amino-terminal sequences of all human CaD isoforms. Exon 4 is spliced in i-CaDs I and h-CaD. Exon 3a is also spliced in all i-CaDs, whereas exon 3ab is specifically spliced in h-CaD. Among these splicing pathways, the most interesting is the regulatory mechanism to select the two consensus sequences for the 5' splice sites in exon 3. The same exon structure as for the human CaD gene has been also found in the genome of chicken CaD (unpublished work). Such use of competing splice sites has been reported in viral transcription units of adenovirus EJA (24), simian virus 40 tumor antigen genes (25), Drosophila Ultrabithorax (26) and transformer genes (27), and the human kininogene gene (28). Among them, the two former examples have been the object of the most study
Not determined
V
Exon sequences are shown in uppercase letters, and intron sequences in lowercase letters. Amino acids encoded by each exon are indicated by one-letter symbols below the nucleotides, and their position numbers at each 5' splice site are from the human aorta h-CaD sequence (22). The sequences and position number with underline are from HeLa I-CaD sequence. I
ttatacao T GTG CCT GAC GAG GAG GCC AAG ACA ACC ACC ACA AAC ACTI CAA E E A V K T T P D T T T N Q
r-----
AG CGC CTG GCT CGG CGT E R L A R R
3a
GAG E
G AT GIG UGAA WUG G AIAT I GAG
E
V
D
G
E
D
A
CGCA TTC CG A F L
GAA AGA CGC CAA AAA CGC CTT CAG GAG GCT CTG GAG CGG CAG AAG GAG TTC GAC CCA E R R Q K R L 0 E A L E R 0 K E F D P
TA ACA GAT GCA AGT CTG TCG CTC CCA AGC AGA AGA ATG CAA AAT GAC ACA GCA GAA AAT GAA ACT ACC GAG AAG GAA T A D S L S L P S R R M 0 N D T A E N E T T E K E AA AGT GAA AGT CGC CAA GAA AGA TAC GAG ATA GAG S E S R E R E K E Q Y
GAA E
284 91
(520)
ACA 365
(601)
AT
3ab
GA" CAI!51A 0 1 V E
GTG
V
ATG m
118
(124)
GAA
446 145
(682) (151)
E
ACA GAA ACA GTC ACC AAG TCC TAC CAG AAG AAT GAT TGG AG T E T V T K S Y Q K N D W R
GTG GAA GAG AAA ACA ACT GAA AGC CAG GAG GAA
V
E
E
K
T
T
E
S
Q
E
E
V
V
M
S
L
K
N
G
Q
ATC AGT TCA GAA GAG CCT AAA CAA GAG GAG GAG AGG GAA CAA GGT TCA GAT GAG ATT TCC CAT CAT GAA AAG ATG GAA GAG S S E E P K Q E E E R E Q G S D E S H E M E H K E GAA GAC AAG GAA AGA GCT GAG GCA GAG AGG GCA AGG TTG GAA GCA GAA GAA AGA GAA AGA ATT AAA GCC GAG CAA GAC AAA E E E A E D K R A R A R E L A E E R E R 0 D K K A E AAG ATA GCA GAT GAA CGA GCA AGA ATT GAA E K A E R A R D
G00 A
GAA GAA AAA GCA GCT GCC CAA GAA AGA GAA AGG AGA GAG GCA GAA GAG E E A K A E R E A R R E E A Q E
AGG GAA AGG ATG AGG GAG GAA GAG AAA AGG GCA SCA GAG E E R E R R E K R A E A M
GAG E
AGG CAG AGG ATA AAG GAG GAA GAG AAA AGG GCA GCA GAG
0
R
R
527 (763) 1T2 (178)
GAh 608 E 1 99 ACA GTG GTA ATG TCA TTA AAA AAT GGG CAG T
K
E
E
E
K
R
A
A
(97)
T
AT GCT GAA GAA AAC AAG AAA GAA GAC AAG GAA AAG GAG GAG GAG GAA GAG GAG AAG CCA AAG CGA GGG AGC ATT GGA A E E N K K E K E K E E E D D E E E K P K R G S G N
12125
E
(844) (205) (925) (232)
(1006)
(259) (1087) (286)
(1 168) (313)
(1249) ( 340)
GAG AGG CAG AGG ATA AAG GAG GAA GAG AAA AGG GCA GCA GAG GAG AGG CAG AGG ATA AAA GAG GAA GAG AAA AGG GCA GCA 0 R E E E E Q R R K K R A E E A R K E E E R A K A
(1330) (367)
GAO E
(1411) (394)
GAA AAG GTA GAA CAG AAA ATA GAA GGG AAA TGG GTA AAT GAA AAG E K V E 0 K G E K W V N E K
(1492) (421)
GAG GAG AGG CAA AGG GCC AGG GCA GAG GAG GAA GAG AAG GCT AAG GTA GAA GAG CAG AAA CGT AAC AAG CAG CTA GAA 0 R A R A E E E E K A K V E E 0 K R N K 0 L E E E R AAA AAA CGT GCC ATG CAA GAG ACA AAG ATA AAA K K R 0 E T K K A M
000 G
AAA GCA CAA GAA GAT AAA CTT CAG ACA GCT GTC CTA AAG AAA 0 E D K L 0 T A V L K K K A
CAaYntacLata Q I
(1537)
(436)
FIG. 4. Nucleotide sequence of exon 3 (uppercase) with flanking intron sequences (lowercase). Exons 3aand 3ab are boxed. The consensus sequence of 5' splice sites for exons 3a and 3ab are underlined, and the intron/exonjunctions are indicated by arrowheads. The nucleotides and the deduced amino acid sequences from HeLa I-CaD cDNAs are numbered at right, and the numbers in parentheses are from human aorta h-CaD cDNA (22).
12126
Proc. Nad. Acad. Sci. USA 89 (1992)
Biochemistry: Hayashi et a!. BarnH n b
94 4
a b
ib
of CaD isoform expression must be studied at both the transcriptional and the mRNA processing level.
01
We thank Dr. T. Hon (National Institute of Radiological Sciences) for his suggestions. This study was partly supported by grants from the Scientific Research Fund of the Ministry of Education, Science, and Culture of Japan and from the Nissan Foundation.
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FIG. 5. Southern blot analysis. Genomic DNA from HeLa S3 (lanes a) and human peripheral lymphocytes (lanes b) was digested with the indicated enzymes. Probes were made from the full-length HeLa I-CaD I cDNA. Size markers are indicated in kilobases.
according to which trans-acting factors might be involved in the modification of the recognition process of alternative 5' splice sites by U1 small nuclear ribonucleoprotein (29, 30). A splicing factor derived from HeLa nuclear extract (SF2; ref. 31) plays a critical role in selection of the 5' splice site; it promotes use of the 5' splice site that is located near the 3' splice site in an artificial mRNA precursor containing two 5' splice sites (32). An anti-SF2 factor to suppress the activation of the 5' splice site by SF2 has been also reported (33). Here we propose the selective usage of competing splice sites in relation to cell differentiation. Regulation of h- and 1-CaD expression may depend on unknown trans-acting factors which are linked to phenotypic modulation of smooth muscle cells. Further studies are required for identification of such factors. Additionally, it is necessary to determine whether HeLa- and WI-38-type mRNAs are transcribed from the same promoter or an independent promoter. The regulation
FIG. 6. Chromosome mapping of human CaD gene. A whole R-banded (pro)metaphase plate was hybridized with the biotinylated CaD gene. Arrows indicate the signals on 7q33-q34.
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