Proc. Natd. Acad. Sci. USA Vol. 89, pp. 12122-12126, December 1992 Biochemistry Genomic structure of the human caldesmon (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 (h- predominantly expressed in differentiated smooth muscle CaD) is predominantly expressed in smooth muscles, whereas cells and is replaced by I-CaD during dedifferentiation (12- the low molecular weight caldesmon (I-CaD) is widely distrib- 14). uted in nonmusce tissues and cells. The changes in CaD To investigate the regulation of CaD isoform expression, isoform expression are closely correlated with the phenotypic we have searched for isoform diversity of human CaDs and modulation ofsmooth muce cells. During a search for isdorm have determined the genomic structure¶ and the chromoso- diversity of human CaDs, I-CaD cDNAs were cloned from mal location ofthe CaD gene. Our studies have revealed two HeLa S3 cells. HeLa i-CaD I is composed of 558 amino acids, splice sites within exon 3 of the CaD gene. We discuss this whereas 26 amino acids (residues 202-227 for HeLa i-CaD I) feature in relation to the regulation of CaD isoform expres- are deleted in HeLa i-CaD H. The short amino i sion. 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 MATERIALS AND METHODS insertion relative to WI-38 I-CaD H. To reveal the mo r Cloning and Sequencing of cDNA. An oligo(dT)-primed events of the expressional regulation of the CaD iorms, the cDNA library from HeLa S3 mRNA was screened with genomic sructure ofthe human CaD gene was detemiu. The 32P-labeled restriction fragments originating from embryonic human CaD gene is composed of 14 exons and was m ed to chicken brain I-CaD cDNA. Four positive clones carrying a single locus, 7q33-q34. The 26-amino acid in is I-CaD cDNAs were obtained and their sequences were de- encoded in exon 4 and Is cay spliced in the mRNAs for termined. both h-CaD and i-CaDs I. Exon 3 is the exon that encodes the Southern Blot Analysis. Genomic DNA (5 pg) from HeLa central repeating domain specific to h-CaD (residues 208-436) S3 cells or human peripheral lymphocytes was digested with together with the common domain in all CaDs (residues 73-207 restriction enzymes and the digests were electrophoresed in for h-CaD and WI-38 i-CaDs, and residues 68-201 for HeLa 0.7% agarose gels. The separated DNA fragments were i-CaDs). The regulation of h- and i-CaD exp is thought blotted to nylon membranes by the method of Southern (15). to depend on selection of the two 5' splice sites within exon 3. The hybridization conditions with 32P-labeled HeLa i-CaD I Thus, the change In essio between I-CaD and h-CaD or II cDNA fragments have been described (9). might be caused by this splicing pathway. Reverse Trauscriptio-PR. The first-strand cDNA from each cell type was synthesized by using (dT)1218 and/or the Caldesmon (CaD), a calmodulin- and actin-binding , antisense primer specific to the 3' noncoding sequence of plays a vital role in the regulation of smooth muscle and human h- and I-CaD cDNAs. Primers used in this experiment nonmuscle contraction (1, 2). Two CaD isoforms have been were as follows: sense primer Pn, d(ATGCTGGGTGGATC- identified; h-CaD (high Mr, 120,000-150,000) and i-CaD (low CGGATC), specific to the short amino-terminal sequence of Mr, 70,000-80,000) asjudged by NaDodSO4/polyacrylamide HeLa I-CaDs; antisense primer Pm, d(GTTTAAGTT- gel electrophoresis (3-6). Sequencing studies on chicken TGTGGGTCATGAATTCTCC), complementary to the com- CaD cDNAs have demonstrated that the deduced molecular mon sequence in all CaD isoforms, nucleotide positions weights ofh- and i-CaD are in the range of87,000-89,000 and 832-859 in WI-38 i-CaD II cDNA; sense primer Pn2, d(CAC- 59,000-60,000, respectively, and that the major parts ofboth CATGGATGATTTTGAGCG), nucleotide positions 108- CaDs have identical amino acid sequences except for the 128 in WI-38 i-CaD II cDNA (16); and antisense primer Pi, insertion of the central repeating domain of the h-CaD d(GAAGGTAGGCTTGTCTTCTTGGAGCTTTTC), com- molecule (7-10). Structural and functional analyses have plementary to the insertion sequence of the HeLa i-CaD I revealed that the calmodulin-, actin-, and tropomyosin- sense strand (Fig. 1). DNA fragments amplified by PCR (17) binding sites contained in a region involved in the regulation were separated in 1.5% agarose gels. of actin-myosin interaction reside within the common car- Cha t a of Human CaD Gene. A human placental boxyl-terminal domain of both CaD isoforms (9, 11). The genomic library in EMBL3 was screened by hybridization tissue and cell distributions of the two isoforms are distinc- with 32P-labeled probes from the HeLa I-CaD I cDNA. tively different, however. h-CaD is primarily found in smooth Restriction mapping revealed four overlapping clones muscles, whereas i-CaD is widely distributed in nonmuscle (EMBL 11, SA, 111, and C4) and a nonoverlapping clone tissues and cells. Notably, the changes in expression of the (EMBL 2) (see Fig. 3A). Restriction fragments from each two CaD isoforms are closely correlated with phenotypic modulation of smooth muscle cells, in which h-CaD is Abbreviations: CaD, caldesmon; h-CaD, high molecular weight CaD; I-CaD, low molecular weight CaD. ITo whom reprint requests should be addressed. The publication costs ofthis article were defrayed in part by page charge IThe nucleotide sequences reported in this paper have been depos- payment. This article must therefore be hereby marked "advertisement" ited in the GenBank/EMBL/DDJB data base (accession nos. in accordance with 18 U.S.C. §1734 solely to indicate this fact. D90452 and D90453). 12122 Downloaded by guest on September 30, 2021 Biochemistry: Hayashi et al. Proc. Natd. Acad. Sci. USA 89 (1992) 12123

0Y clone were subcloned and sequenced. Clones carrying the C5~ 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 sense Pri Pn2 Pil Pn2 improvement of the specific-primer PCR method (18, 19). antisense Prn Pm Pi Pi The specific DNA fragment containing the target exon thus obtained was used for isolation ofthe genomic clone (EMBL F5). ) 731 752__ _~af 691 Chromosomal Mapping of Human CaD Gene. The human -670 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). FIG. 2. Characterization ofI-CaD isoforms expressed in HeLa S3 RESULTS and WI-38 cells. The reverse transcription-PCR method was done with the indicated primers sets, using first-strand cDNA from HeLa Isoform Diversity ofHuman CaDs. Sequence analysis in the S3 and WI-38 as templates. Sizes ofthe amplifiedfragments are given present study revealed the two different molecules of i-CaD in base pairs. (i-CaDs I and II) that originated from HeLa S3 cells. The primary structures of HeLa I-CaDs I and II in comparison using a HeLa-type sense primer (Pn) and the common with those ofW138 i-CaD II (16) and human aorta h-CaD (22) antisense primer (Pm). The similarly amplified DNA frag- are shown schematically in Fig. 1. HeLa i-CaD I and II are ments (752 and 830 bp) were obtained from WI-38 mRNA by composed of558 amino acids (Mr 64,252) and 532 amino acids using a WI-38-type sense primer (Pn2). The result suggests (Mr of 61,210), respectively; 26 amino acids (residues 202- that the two i-CaD isoforms are expressed in HeLa S3 and 227) of HeLa i-CaD I have been deleted in HeLa i-CaD II. WI-38 cells. In both cases, large and small DNA fragments The short amino-terminal sequences (residues 1-18) ofHeLa would be derived from the mRNAs for the respective i-CaD i-CaDs are different from those of WI-38 i-CaD II and aorta with the insertion of 26 amino acids (i-CaD I) and without it h-CaD (residues 1-24). The 26-amino acid insertion in HeLa (i-CaD II). Large DNA fragments were not well amplified, i-CaD I is found in aorta h-CaD, but not in WI38 i-CaD II. The however. Immunoblotting of HeLa S3 and WI-38 cells re- central repeating domain specific to h-CaD (residues 208- vealed that the expression of I-CaD I was very low compared 436) is deleted in all i-CaDs. To search for isoform diversity with that of I-CaD II (data not shown). Therefore, such of human CaD, the reverse transcription-PCR method was amplifications would be reflected in the amount of each introduced (Fig. 2). The primers used in this experiment are mRNA for I-CaD I or II. The PCR with an antisense primer indicated in Fig. 1. The two kinds ofDNA fragments [731 and specific to the HeLa I-CaD I insertion sequence (Pi) could 809 base pairs (bp)] were amplified from HeLa S3 mRNA by clearly amplify a single fragment- 670- and 691-bp fragments

HeLa I-CaD - Pm 558 amino acids MLGGSGSHGRRSLAALSQ GEEKGTKVQAKREKLQEDKPTFKKEE 1 18 202 - Pi 227

HeLa I-CaD 11 532 amino acids

MLGGSGSHGRRSLAALSQ 1 18

WI-38 I-CaD 11 538 amino acids .M MDDFERRRELRRQKREEMRLEAER 1 24 Pn2 -

WI-38 I-CaD 564 amino acids

MDDFERRRELRRQKREEMRLEAER GEEKGTKVQAKREKLQEDKPTFKKEE 1 24 208 233

aorta h-CaD _ _ 793 amino acids

MDDFERRRELRRQKREEMRLEAER GEEKGTKVQAKREKLQEDKPTFKKEE 1 24 437 462

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. Downloaded by guest on September 30, 2021 12124 Biochemistry: Hayashi et al. Proc. Nad. Acad. Sci. USA 89 (1992) from HeLa S3 and WI-38 mRNAs, respectively. From these h-CaD. Exon 3a is spliced in the mRNAs for all i-CaDs, results, we have identified WI-38 i-CaD I with the insertion whereas exon 3ab is specifically spliced in the mRNA for sequence in WI-38 cells (Fig. 1). The primary structures of h-CaD. Exons 1 and 1' encode the short amino terminus the human CaD isoforms identified are summarized in Fig. 1. specific to HeLa i-CaDs and to WI-38 I-CaDs or aorta h-CaD, Structure of Human CaD Gene. Five positive clones were respectively. isolated from a human placental genomic library. Each clone Chromosomal Locus of Human CaD Gene. Southern blot was subjected to further characterization, and the exon- analysis of genomic DNA from HeLa S3 cells and human containing fragments derivedfrom the respective clones were peripheral lymphocytes with HeLa I-CaD I cDNA fragments subcloned and sequenced. Fig. 3A shows the genomic con- as probes revealed identical hybridizing patterns (Fig. 5). The struction ofhuman CaD. Four overlapping clones (EMBL 11, same result was obtained with HeLa i-CaD II cDNA fiag- 5A, 111, and C4) carried most of the exons. EMBL 2 and ments as probes (data not shown). These results suggest that EMBL F5 were independent clones carrying an exon encod- the CaD isoforms are encoded by a single gene. To confirm ing the short amino-terminal sequence specific to HeLa this suggestion, the chromosomal locus ofthe CaD gene was i-CaDs (residues 6-17) or WI-38 I-CaDs and aorta h-CaD determined. We examined 100 (pro)metaphase plates show- (residues 1-24). Since EMBL 2 and EMBL F5 did not overlap ing a typical R-band for all clones. The efficiency of hybrid- with EMBL 11, we could not clarify the spatial relationship ization was similar among the four kinds ofprobe (EMBL 2, between exon 1 and 1'. All intron/exon junctions (Table 1) 11, 111, and C4, indicated in Fig. 3A), and the locations ofthe are compatible with the splice consensus sequence except for signals were the same. For example, 51% of such R-banded exon 3 (23). exhibited complete double-spot staining with Exon 3 is constituted as follows (Fig. 4). The common EMBL 11 as a probe. The signals were localized in band domain of the CaD isoforms (residues 68-201 for HeLa q33-q34 of the long arm of 7. No signals were i-CaDs and 73-207 for WI-38 I-CaDs and aorta h-CaD) is detected in the other chromosomes. Thus, the CaD gene encoded in exon 3a, whereas the central repeating domain could be assigned to band 7q33-q34 (Fig. 6). 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 DISCUSSION Fig. 4). Exon 4 encodes the insertion sequence specific to the In our previous study (9), we investigated the structural and two i-CaDs I and aorta h-CaD. Based on the present findings, functional relationships between chicken h- and I-CaDs, in alternative splicing pathways are summarized in Fig. 3B. which the major parts of the amino and carboxyl termini of Exons 2 and 5-13 are spliced in all of the mRNAs for h- and both isoforms are completely identical sequences. The car- i-CaDs, and exon 4 is spliced in the mRNA for I-CaDs I and boxyl terminus of both chicken CaDs conserve two se-

A FAMB[L. EMB. F;.-. L-MBL

,Akr..a.jj :.1-I'. ii II 1 j 1 ii 1i

L..:~ ~ H-, L... t.1 ..1.i

II .-.U,r- 1 .1 ..IVI.. 1.1... ..i.

I.

o..1 -ll ......

;J D.fi _

k-iirTaar±ortasrriootrl

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. Downloaded by guest on September 30, 2021 Biochemistry: Hayashi et al. Proc. Natl. Acad. Sci. USA 89 (1992) 12125 Table 1. Exon organization of human CaD gene quences showing high homology with the two tropomyosin- Size, binding regions (T1 and T2) in the troponin T molecule. We 3' splice site Exon bp 5' splice site have further identified the minimum regulatory domains, which are involved in the Ca2+-dependent regulation of the Not determined 1 >37 CGCTCTCCCAgtgagt actin-myosin interaction, in the carboxyl terminus ofchicken AL r tttcagGTCCAGACAT 1' 112 AAGCAGAAAGgtaagg h- and i-CaDs. We compared the primary structures ofhuman noncoding E A E23 CaDs with those of chicken CaDs. The overall sequence ttgoagAATCGCCTAC 2 147 CCCAGAACAGgtactg identity between the two species of CaD is 65-68%. How- R I A Y A Q N72 ever, all CaD isoforms in different species strongly conserve Q the minimum regulatory domain (89% identity). In addition to gtacagTGTGCCTGAC 3 1090 AAAGAAACAGgtacag this, the amino termini of all CaD isoforms (for example, S V P D L K K Q436 residues 21-47 and 85-121 for HeLa I-CaD I and the corre- aaaaagGGAGAAGAGA 4 78 AAAAGAAGAGgtaaat sponding residues of other chicken and human CaDs) also G E E K K E E42 retain completely identical sequences. Therefore, these con- ggttagATCAAAGAEG 5 146 ATACTTTCAGgtaaga served domains might be important for the structure and I K D N T F510 function of CaDs. ccacagCCGCCCTGGA 6 264 CAGAGAGGAGgtaagg To begin to elucidate the molecular events ofthe regulation S R D G L R E E598 of CaD isoform expression during phenotypic modulation of tcttagGAAGAGAAGA 7 141 ATCTCTCAAGgtattt smooth muscle cells, we have investigated the genomic E E K S S L K65 structure ofthe human CaD gene. The CaD gene is composed tcotagATAGAAGAGC 8 44 TGCAGAAAAGgtaaat of at least 14 exons (Fig. 3) and was mapped to a single locus I E E V Q K659 on 7q33-q34 by using the four kinds ofprobes that can cover ttttagCAGTGGTGTC 9 82 TGCAATTGAGgtgaga the overall CaD gene (Fig. 6). The isoform diversity of this S S G V S A I E687 protein (Fig. 1) can be explained by selection of exon 1 or 1', tttcagGGAACAAAAA 10 138 ACCAAATAAGgtgagc exon 3a or 3ab, and/or exon 4. Exon 1 or 1' encodes the short G T K T P N K733 amino-terminal sequences of all human CaD isoforms. Exon ttgtagGAAACTGCTG 11 96 CAAACCTTCTgtaagt 4 is spliced in i-CaDs I and h-CaD. Exon 3a is also spliced in E T A P K P S765 all i-CaDs, whereas exon 3ab is specifically spliced in h-CaD. tatcagGACTTGAGAC 12 81 CCCCACTAAGgtaatc Among these splicing pathways, the most interesting is the D L R S P T K792 regulatory mechanism to select the two consensus sequences tggoagGTTTGAGACG 13 >533 Not determined for the 5' splice sites in exon 3. The same exon structure as V for the human CaD gene has been also found in the genome Exon sequences are shown in uppercase letters, and intron se- of chicken CaD (unpublished work). Such use of competing quences in lowercase letters. Amino acids encoded by each exon are splice sites has been reported in viral transcription units of indicated by one-letter symbols below the nucleotides, and their adenovirus EJA (24), simian virus 40 tumor antigen position numbers at each 5' splice site are from the human aorta (25), Drosophila Ultrabithorax (26) and transformer genes h-CaD sequence (22). The sequences and position number with (27), and the human kininogene gene (28). Among them, the underline are from HeLa I-CaD sequence. two former examples have been the object of the most study

ttatacao T GTG CCT GAC GAG GAG GCC AAG ACA ACC ACC ACA AAC ACTI CAA GIG UGAA WUG GAIATG ATI GAG CGCA TTC CG 284 (520) I r----- V P D E E A K TT TT N T Q V E G DD E AA F L 91 (97) AG CGC CTG GCT CGG CGT GAG GAA AGA CGC CAA AAA CGC CTT CAG GAG GCT CTG GAG CGG CAG AAG GAG TTC GAC CCA ACA 365 (601) E R L A R R E E R R Q K R L 0 E A L E R 0 K E F D P T 118 (124) 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 GAA 446 (682) T D A S L S L P S R R M 0 N D T A E N E T T E K E 145 (151) 3a E AA AGT GAA AGT CGC CAA GAA AGA TAC GAG ATA GAG GAA ACA GAA ACA GTC ACC AAG TCC TAC CAG AAG AAT GAT TGG AG 527 (763) K S E S R Q E R Y E E E T E T V T K S Y Q K N D W R 1T2 (178) 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 GAh 608 (844) D A E E N KK E D K E K E E E E E E K P K R G S G E 1 99 (205) AT CAI!51A GA" GTG ATG GTG GAA GAG AAA ACA ACT GAA AGC CAG GAG GAA ACA GTG GTA ATG TCA TTA AAA AAT GGG CAG (925) N 0 1 V E V m V E E K T T E S Q E E T V V M S L K N G Q (232) 3ab 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 (1006) S S EE P K Q E E E R E Q G S D E S H H E K M E E (259) 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 (1087) E D K E R A E A E R A R L E A E E R E R K A E 0 D K (286) AAG ATA GCA GAT GAA CGA GCA AGA ATT GAA G00 GAA GAA AAA GCA GCT GCC CAA GAA AGA GAA AGG AGA GAG GCA GAA GAG (1 168) K A D E R A R E A E E K A A A Q E R E R R E A E E (313) AGG GAA AGG ATG AGG GAG GAA GAG AAA AGG GCA SCA GAG GAG AGG CAG AGG ATA AAG GAG GAA GAG AAA AGG GCA GCA GAG (1249) R E R M R E E E K R A A E E R 0 R K EEE K R A A E (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 (1330) E R 0 R K E E E K R A A EE R Q R K E E E K R A A (367) 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 GAO (1411) E E R 0 R A R A E E E E K A K V E E 0 K R N K 0 L E E (394) AAA AAA CGT GCC ATG CAA GAG ACA AAG ATA AAA 000 GAA AAG GTA GAA CAG AAA ATA GAA GGG AAA TGG GTA AAT GAA AAG (1492) K K R A M 0 E TK K G E K V E 0 K E G K W V N E K (421) AAA GCA CAA GAA GAT AAA CTT CAG ACA GCT GTC CTA AAG AAA CAaYntacLata (1537) K A 0 E D K L 0 T A V L K K Q I (436) FIG. 4. Nucleotide sequence ofexon 3 (uppercase) with flanking intron sequences (lowercase). Exons 3aand 3ab are boxed. The consensus sequence of5' 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). Downloaded by guest on September 30, 2021 12126 Biochemistry: Hayashi et a!. Proc. Nad. Acad. Sci. USA 89 (1992) BarnH EcoR Hind ill of CaD isoform expression must be studied at both the n b a b ib transcriptional and the mRNA processing level. We thank Dr. T. Hon (National Institute ofRadiological Sciences) 23.1 A, for his suggestions. This study was partly supported by grants from the Scientific Research Fund of the Ministry of Education, Science, 94 01 6 6 ~- and Culture of Japan and from the Nissan Foundation. 4 4~ Itd 2 3 1. Sobue, K., Muramoto, Y., Fujita, M. & Kakiuchi, S. (1981) 2.0 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, 3133- 3137. 4. Sobue, K., Tanaka, T., Kanda, K., Ashino, N. & Kakiuchi, S. (1985) Proc. Natl. Acad. Sci. USA 82, 5025-5029. FIG. 5. Southern blot analysis. Genomic DNA from HeLa S3 5. Bretcher, A. & Lynch, W. (1985) J. Cell Riol. 100, 1748-1757. (lanes a) and human peripheral lymphocytes (lanes b) was digested 6. Dingus, J., How, S. & Bryan, J. (1986) J. Cell Biol. 102, with the indicated enzymes. Probes were made from the full-length 1748-1757. HeLa I-CaD I cDNA. Size markers are indicated in kilobases. 7. Hayashi, K., Kanda, K., Kimizuka, F., Kato, I. & Sobue, K. (1989) Biochem. Biophys. Res. Commun. 164, 503-511. according to which trans-acting factors might be involved in 8. Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R. G. & Lin, the modification of the recognition process of alternative 5' W.-G. (1989) J. Biol. Chem. 264, 13873-13879. 9. Hayashi, K., Fujio, Y., Kato, I. & Sobue, K. (1991) J. Biol. splice sites by U1 small nuclear ribonucleoprotein (29, 30). A Chem. 266, 355-361. splicing factor derived from HeLa nuclear extract (SF2; ref. 10. Bryan, J. & Bryan, L. (1991) J. Muscle Cell Motil. 12, 372-375. 31) plays a critical role in selection of the 5' splice site; it 11. Wang, C.-L. A., Wang, L.-W. C., Xu, S., Lu, R. C., Saavedra- promotes use of the 5' splice site that is located near the 3' Alanis, V. & Bryan, J. (1991) J. Biol. Chem. 266, 9166-9172. splice site in an artificial mRNA precursor containing two 5' 12. Ueki, N., Sobue, K., Kanda, K., Hada, T. & Higashino, K. splice sites (32). An anti-SF2 factor to suppress the activation (1987) Proc. Natl. Acad. Sci. USA 84, 9049-9053. 13. Sobue, K., Kanda, K., Tanaka, T. & Ueki, N. (1988) J. Cell. of the 5' splice site by SF2 has been also reported (33). Here Biochem. 37, 317-325. we propose the selective usage of competing splice sites in 14. Glukhova, M. A., Kabakov, A. E., Frid, M. G., Ornatsky, relation to cell differentiation. Regulation of h- and 1-CaD 0. I., Belkin, A. M., Mukhin, D. N., Orekhov, A. N., Kotel- expression may depend on unknown trans-acting factors iansky, V. E. & Smirnov, V. N. (1988) Proc. Natl. Acad. Sci. which are linked to phenotypic modulation ofsmooth muscle USA 85, 9542-9546. cells. Further studies are required for identification of such 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. factors. Additionally, it is necessary to determine whether Chem. 266, 16917-16924. HeLa- and WI-38-type mRNAs are transcribed from the 17. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K., Horn, G. T., same promoter or an independent promoter. The regulation 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. & Mc- Keown, 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. FIG. 6. Chromosome mapping of human CaD gene. A whole 32. Krainer, A. R., Conway, G. C. & Kozak, D. (1990) Cell 62, R-banded (pro)metaphase plate was hybridized with the biotinylated 35-42. CaD gene. Arrows indicate the signals on 7q33-q34. 33. Mayeda, A. & Krainer, A. R. (1992) Cell 68, 365-375. Downloaded by guest on September 30, 2021