Proc. Nati. Acad. Sci. USA Vol. 88, pp. 10811-10815, December 1991
Cell Biology
Structure of the human villin gene (actin-binding protein/gene evolution/intestinal differentiation)
ERIC PRINGAULT, SYLVIE ROBINE,
AND
DANIEL LOUVARD
Unite de Biologie des Membranes, Centre National de la Recherche Scientifique, Unitt Associde 1149, DIpartement de Biologie Molculaire, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France
Communicated by Frangois Jacob, August 26, 1991 (received for review July 12, 1991)
ABSTRACT We have isolated and characterized the complete human villin gene. The villin gene is located on chromosome 2q35-36 in humans and on chromosome 1 in mice. Villin belongs to a family of calcium-regulated actin-binding proteins that share structural and functional homologies. The villin gene is expressed mainly in cells that develop a brush border, such as mucosal cells of the small and large intestine and epithelial cells of the kidney proximal tubules. Villin gene expression is strictly regulated during adult life and embryonic development in the digestive and urogenital tracts and, thus, may be used as a marker of the digestive and renal cell lineages. The human villin gene has one copy per haploid genome, encompasses about 25 kilobases, and contains 19 exons. Analysis of the structural organization of this gene shows that the two mRNAs that encode villin in humans arise by alternative choice of one of the two polyadenylylation signals located within the last exon. The overall organization of the exons reflects the gene duplication event from which this family of actin-binding proteins originated.
structural and functional organization of the protein sequence. We suggest that the two transcripts observed in humans arise from a common pre-mRNA by the random choice of one of two polyadenylylation signals located in the last exon.
MATERIALS AND METHODS Southern Blots. Human genomic DNA was purified from lymphocytes and digested to completion with various restriction enzymes, subjected to electrophoresis, and blotted onto nitrocellulose membranes as described by Southern (16). Blots were then hybridized with three different villin cDNA probes labeled with [a-32P]dCTP by nick-translation. Genomic Library Screening. A genomic library (a gift of H. Lehrach, European Molecular Biology Laboratory, Heidelberg), constructed in phage A by cloning partially Sau3A1digested human lymphocyte genomic DNA into the BamHI sites of the EMBL3 vector (17) was screened with a mixture of overlapping villin cDNA probes spanning the entire length of the mRNA (3.5 kb). Positive clones were then rescreened separately with each probe. Two overlapping clones were selected for further analysis: EMBL3-85e contains the 5' end of the gene, and EMBL3-30a contains the 3' end. DNA Amplification (PCR). Amplification of fragments of the villin gene was performed using the Perkin-Elmer/Cetus thermal cycler and cloned Taq polymerase (Cetus). The temperature cycle was 920C, 1 min; 550C, 1 min; 720C, 3 min (with an increase in elongation time of 2 sec per cycle). The number of cycles was 30-40 depending of the experiment. DNA Sequencing. Exons and the junctions between exons and intervening sequences were sequenced (18) directly on DNA purified from phage EMBL3-85e or -30a. Specific oligonucleotide primers chosen from the coding sequence of the villin cDNA were used. The complete villin cDNA sequence has already been published (13) and has been deposited in the EMBL/GenBank data base (HSVILLR). S1 Nuclease Mapping. A 380-base single-stranded DNA probe spanning the 5' end of the villin mRNA was generated by synthesizing the noncoding strand of a genomic restriction fragment subcloned in the bacteriophage M13 mpl8 with Klenow enzyme, from an oligonucleotide primer located in the first exon of the gene. This probe was labeled by incorporating [a-32P]dCTP during synthesis and purified by electrophoresis in a 6% polyacrylamide gel containing 7 M urea. An aliquot of the probe (10,000 cpm) was hybridized at 48°C for 16 hr in the presence of 10 ,ug of total RNA from either HeLa cells or HT-29 differentiated cells, in 80% formamide/40 mM Pipes/400 mM NaCl/1 mM EDTA with pH adjusted to 6.4. The heteroduplex hybrids were then digested for 1 hr by S1 nuclease (Pharmacia) at concentrations ranging from 0 to 1000 units/ml in 30 mM NaOAc, pH 4.4/280 mM NaCI/4.5 mM Zn(OAc)2 containing salmon
The intestinal mucosa provides an attractive model with which to study the physiological role and the regulation of expression of tissue-specific genes. Arising from stem cells located in the intestinal crypts, immature precursors of enterocytes migrate toward the tips of villi, gradually acquiring their differentiated phenotype. This crypt/villus spatiotemporal gradient of differentiation allows us to establish correlations between the expression of specific proteins and the morphological maturation of enterocytes. One of these proteins, villin, is of particular interest since (i) it belongs to a family of actin-binding proteins that are modulated by calcium (1-6), (ii) it plays a key role in the assembly of the brush border (7), (iii) it is expressed in immature precursors of enterocytes (8-12), and (iv) it displays a strict tissuespecific distribution (8). We previously isolated and characterized a complete cDNA clone coding for human villin (13). The primary structure of villin displays a large duplicated domain common to other actin-severing proteins and a specific additional domain necessary for bundling. Expression of villin mRNA has been studied in various tissues from different species (14). In humans, there are two villin mRNA transcripts; the characterization of the corresponding cDNAs has shown that these two mRNAs differ by an extension of 800 bases in the 3' noncoding region (13). The villin gene maps to human chromosome 2q35-36 and mouse chromosome 1 and belongs to a cluster of genes conserved between the two species (15). Here we report the cloning and characterization of the complete human villin gene. There is one copy of the villin gene per human haploid genome. This gene encompasses about 25 kilobases (kb) and contains 19 exons. The general distribution of exons and intervening sequences reflects the 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.
Abbreviation: nt, nucleotide(s). 10811
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sperm DNA at 50 Ag/ml. Protected fragments were analyzed in an 8% polyacrylamide gel containing 7 M urea.
RESULTS AND DISCUSSION Human Haploid Genome Contains One Copy of the Vilhin Gene. Previous in situ hybridization experiments with a villin probe on human chromosomes have provided evidence for the existence of a single locus for the villin gene, on chromosome 2, sub-band q35-36 (15). As there are two villin mRNAs in humans, we used Southern hybridization to examine the number of villin gene copies present at this locus. Total genomic human DNA was digested with various restriction enzymes (EcoRI, BamHI, Bgl II, or combinations), blotted onto nitrocellulose (16), and hybridized with a probe spanning the 5' end of the villin gene from nucleotide (nt) -300 to nt +80 (Fig. 1). This probe contains no restriction sites for the enzymes used. Autoradiography revealed only one hybridizing restriction fragment for each enzyme tested. The experiment was repeated with two different villin cDNA probes, corresponding either to the amino terminus or to the carboxyl terminus of the molecule, giving similar results (data not shown). These data demonstrate that there is only one copy of the villin gene per human haploid genome and that the two mRNAs are therefore transcribed from the same gene. Isolation of the Human Villin Gene. The previously isolated human villin cDNA (13) was used to screen a bacteriophage library of human genomic DNA in the vector EMBL3 (17). After -4 human genome equivalents were screened with a mixture of overlapping probes covering the entire villin cDNA, 12 positive phages were isolated and purified. These positive phages were subjected to restriction enzyme digestion and Southern blotting using either 5' or 3' cDNA probes (data not shown). Two phages containing overlapping inserts, EMBL3-85e and EMBL3-30a, corresponding to the 5' and the 3' regions of the cDNA, respectively, were analyzed in detail. EMBL3-85e contained an insert of about 15 kb, of which 2 kb was 5' flanking sequence and 13 kb was from the 5' region of the villin gene. EMBL3-30a contained an insert of 19 kb, consisting of 13 kb of the 3' region of the villin gene and about 6 kb of 3' flanking sequence. Structure of the Human VilWin Gene. PCR DNA amplification combined with DNA sequence analysis allowed us to elucidate the exon/intron structure of the villin gene. DNA amplification from genomic clones using primers located in 1
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the cDNA allow one to test for the presence or absence of introns by comparing the length of the amplified fragment with the distance separating the two primers on the cDNA. The difference in size between these fragments should represent the size of the intervening sequence. The exact position of each exon/intron boundary was then determined by sequencing the entire coding sequence and a portion of the flanking introns directly from phage DNA containing the villin gene, using coding oligonucleotides as primers. Exon sequences determined from the villin genomic fragments were consistently identical to the villin cDNA sequence previously obtained from a human colon cell line (13). Fig. 2 summarizes the results obtained using this strategy and shows amplified DNA fragments obtained using primers bordering introns 13 and 14. Genomic DNA was amplified using a 5' coding primer beginning at nt 1604 and a 3' noncoding primer ending at nt 1749 of the villin cDNA. The predicted length of coding sequence between these two primers is 145 bp whereas the size of the corresponding genomic fragment was about 850 bp. The difference of about 700 bp represents the length of intervening sequences present between nt 1604 and 1749 of the cDNA. To determine whether these intervening sequences were distributed in one or more introns, the coding sequence and exon/intron boundaries were elucidated in this region of the genomic clone. We found that the intervening sequence of 700 bp represents a single intron located between nt 1702 and 1703 of the cDNA (numbered as intron 13 once the entire gene structure was determined). When the same 5' primer was used with a different 3' primer ending at nt 1849 on the cDNA (predicting a coding sequence of 245 bp), the size of the amplified genomic fragment was about 950 bp, confirming that the length of intervening sequence in this region is about 700 bp and indicating that there are no introns between nt 1749 and 1849. Another example is shown in lane 3, where predicted length of the coding sequence is 200 bp whereas the primers used amplified a genomic fragment of about 1600 bp, suggesting an intervening sequence of about 1400 bp between nt 1794 and nt 1994. Sequence analysis of the genomic fragment
0 2
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FIG. 1. Hybridization analysis of genomic fragments from the human villin gene. Aliquots of human genomic DNA (15 ,ug) were digested to completion with EcoRI (lane 1), BamHI (lane 2), BgI II (lane 3), EcoRI/BamHI (lane 4), and EcoRI/Bgl II (lane 5). Restriction fragments were fractionated in a 0.8% agarose gel, denatured, transferred onto nitrocellulose, and hybridized with a partial villin cDNA probe (300 bp) labeled with [a-32P]dCTP by nick-translation.
FIG. 2. Mapping of intervening sequences in the human villin gene by genomic DNA amplification (PCR). Villin gene fragments were amplified from 1 ng of EMBL3-30a phage DNA, using several pairs of primers from villin cDNA, by the Taq polymerase chain reaction. Results of only three amplification experiments are shown. A DNA digests (Boehringer Mannheim) were used as molecular size markers. C, no-DNA control. Lanes 1, amplification with 5' primer beginning at nt 1604 and 3' primer ending at nt 1749 of the cDNA; lanes 2, amplification with 5' primer beginning at nt 1604 and 3' primer ending at nt 1849 of the cDNA; lanes 3, amplification with 5' primer beginning at nt 1794 and 3' primer ending at nt 1994 of the mRNA.
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Biology: Pringault et al.
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Proc. Natl. Acad. Sci. USA 88 (1991) ?
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FIG. 3. Exon/intron organization ofthe human villin gene. Exons are represented by solid boxes numbered 1 to 19 from 5' to 3' in the direction of transcription. The connecting line represents intervening sequences. Question marks indicate three intervening sequences for which DNA amplification products of the intron could not be obtained using flanking primers and thus should be longer than 3 kb. The two EMBL3 phages containing villin genomic DNA that were analyzed in detail are represented below the gene.
showed that this intervening sequence represented a single intron located between nt 1848 and nt 1849 of the cDNA (intron 14). Proceeding along the entire villin gene, we mapped all the intervening sequences by this method. No PCR amplification of fragments longer than 2.5 kb was obtained, and therefore the absence of amplification products suggested the presence of introns of 3 kb or larger. The precise size of these huge introns could not be determined by this method and was not investigated further. Fig. 3 shows the general exon/intron organization of the human villin gene determined by detailed analysis of the two genomic fragments contained in phage (EMBL3-85e and EMBL3-30a). The villin gene encompasses 25 kb and is interrupted by 18 introns ranging in size from 83 bp to 3 kb or larger, such as introns 6, 8, and 9, which yielded no amplified fragments in PCR using flanking primers. Thus the human villin gene contains a total of 19 exons including 18 short exons of 79-203 bp and one larger exon (no. 19) of 1041 bp (Table 1). Sequences of the exon/intron boundaries are shown in Table 2. In all cases, the first two bases of the introns are GT, and the last two bases are AG, in agreement with the consensus sequence of donor and acceptor splice sites. Transcription Initiation Site. To map the site of initiation of villin transcription, an antisense genomic probe overlapping the ATG translation start site of the villin gene was hybridized with total RNA from the intestinal cell line CaCO2 and the heteroduplex was digested with S1 nuclease. Protected fragments were then analyzed in an acrylamide gel (data not shown). Control for the specificity of protection was carried out with total RNA isolated from HeLa cells, which do not Table 1. Exon positions and sizes Exon Position in mRNA, nt*
Size, bp
1-97 97 75 98-172 197 173-369 370-478 109 S 111 479-589 203 6 590-792 7 793-871 79 8 872-970 99 971-1124 154 9 1125-1225 101 10 11 1226-1363 138 12 1364-1522 159 13 1523-1702 180 14 1703-1848 146 15 145 1849-1993 16 1994-2182 189 17 2183-2251 69 141 18 2252-2392 19 2393-3434 1041 *Numbered 5' to 3' in the direction of transcription. 1
2 3 4
produce villin mRNA. A single fragment was protected from digestion by total RNA isolated from Caco-2 cells but not by total RNA from HeLa cells. The length of the protected fragment was determined by comparison with a sequence ladder from the genomic fragment, obtained using the primer used to synthesize the probe. This allowed us to determine directly the position of the first protected nucleotide. The single product extended 20 nt upstream of the translation initiation site, to the bold underlined cytosine residue of the sequence CCTTCTCCCCCAGGCTCACTCACCATGACC. Overexposure of the gel did not reveal any additional protected fragments (data not shown). This experiment cannot exclude the possibility that an intron longer than 300 bp located just upstream from this cytosine residue could be responsible for the length of the observed protected fragment. Primer extension experiments were unsuccessful, possibly due to the presence of secondary structure on the 5' end of the mRNA. This difficulty was previously encountered when cloning the 5' end of the villin cDNA (13) and has also been reported in the case of the gelsolin gene (19). Nevertheless, the presence of the translation start site at this position can be argued to be due to the absence of an AG splice acceptor site just upstream of the cytosine residue, a feature that appears to be the rule in all introns of the villin gene. Moreover, we have isolated and characterized 2 kb of5' flanking sequence containing putative Table 2. Intron sizes and exon/intron boundaries 3' acceptor 5' donor Intron Size 1 450 ATC GAG gtga ..... ttag GCC ATG 2 1400 CTG GCT gtga ..... ctag ATC CAC 3 700 CTT GT gtag ..... tcag G ATC 4 150 GGA GAG gtag ..... ctag GTA GAG 5 CTC AGG gtaa ..... gcag GGC ATG 1000 6 TAC CA gtga .... . acag T GTG >3000? 7 150 CAC GAG gtaa ..... ctag GAC TGT 8 >3000? GCG CTG gtgt ..... ccag AAC TTC 9 1000 GTG G gtga ..... ctag CC AAA 10 83 GTG CAG gtat ..... ctag GTG TGG 11 600 TGG CAG gtca ..... ccag GGC AGC 12 TAC CAG gtgt ..... gcag GGG GGC 1750 13 700 GGG AAG gtgt ..... ctag GGT TGT 14 1400 AAG AG gtaa ..... gcag A CTA 15 GAC CAG gtag ..... atag GTC TTC 500 16 1500 TGG AGT gtga ..... gcag AAC ACC 17 >3000? ACT GCT gtga ..... ctag GAG GTC 18 900 AAG GAG gtag ..... ctag GAG CAC Introns are numbered from 5' to 3' in the direction of transcription. Approximative sizes of introns determined by DNA amplification are shown. Question marks show three intervening sequence sizes for which DNA amplification products of the intron were never obtained using flanking primers, indicating that the intron is longer than 3 kb (see Results). Splice 5' donor and 3' acceptor sites are also shown. Exon sequences are in uppercase letters. Intron sequences are in lowercase letters. Nucleotides corresponding to the splicing consensus site are in bold letters.
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Proc. Natl. Acad. Sci. USA 88 (1991)
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FIG. 4. Transcription of the two mRNAs from the human villin gene. (Top) Structural organization of villin, represented from the amino terminus to the carboxyl terminus of the protein, showing the large repeated domain (open boxes), homologous to other severing proteins and containing repeated elements (dashed and black boxes), and the specific domain (HP) (see ref. 13). (Middle) The two mRNAs are depicted by horizontal lines. Positions of translation initiation codons (ATG), stop codons (TGA), polyadenylylation signals (AATAA or AATAAA), and poly(A) extensions are indicated. (Bottom) Schematic representation of the structure of the human villin gene. Exons are represented by solid boxes numbered 1 to 19 from 5' to 3' in the direction of transcription. The thin lines connecting one exon to the next represent intervening sequences drawn to scale. Question marks illustrate intervening sequences greater than 3 kb.
upstream transcription factor-responsive elements that promote in vitro transcription of a reporter gene when transfected into cells expressing villin, but not when transfected into villin-negative cells (unpublished observations). Two mRNAs from the Villin Gene. Northern blot hybridization studies have revealed the presence of two mRNA transcripts (2.7 and 3.5 kb) encoding villin in humans (14). By cloning and sequencing the corresponding cDNAs, we have demonstrated that this size difference is entirely due to an
800-nt extension of the 3' noncoding region in the larger mRNA and that coding sequences are identical in the two mRNAs (13). Thus, it is likely that the two mRNAs do not encode different villin protein isoforms. The villin gene appears to have a single transcription start site located 21 nt upstream from the ATG initiation codon, suggesting that these two mRNAs arise from a single precursor. The 3' noncoding region of the larger mRNA must contain the two polyadenylylation signals used to generate the two mRNAs.
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FIG. 5. Intron/exon distribution of the human villin gene compared to structural and functional domains of the protein. (A) Schematic diagrams of the primary structure of villin (U~pper) and of the exon distribution in the human gene (Lower) are aligned. In the protein, large open boxes represent the duplicated domains containing internal repeats (hatched and black boxes). The specific carboxyl-terminal domain is represented by a stippled box (HP). Coding exons are represented by light or dark stippled boxes, whereas 3' noncoding sequence in the last exon is shown by an open box. (B) Exon distributions in the two duplicated domains of the villin gene are aligned.
Cell
Biology: Pringault et al.
Here we demonstrate that the two polyadenylylation signals are located in the last exon (no. 19) of the villin gene (Fig. 4). The two mRNAs differing in size in the 3' noncoding region may therefore be generated by alternative choice of polyadenylylation signal rather than by alternative splicing of exons. Finally, since the two villin mRNAs are expressed in equimolar ratio in all villin-positive tissues (normal and tumor) so far analyzed, and since preferential expression of one transcript has not been observed with respect to the stage of differentiation of intestinal cells or in any cells able to produce villin (14), it is legitimate to suggest that the site of polyadenylylation is randomly selected. The physiological significance of an alternative 3' noncoding extension of villin mRNA is not known. Evolution of the Villin Gene. Villin belongs to a family of four known proteins that share F-actin-severing activity and amino acid sequence homologies (13). These are fragmin and severin, isolated from lower eukaryotes, and gelsolin and villin, found in higher eukaryotes (20-22). These proteins share a large structural domain containing highly conserved repeated motifs, and all four proteins display an F-actinsevering activity. There is one copy of this structural domain in fragmin and severin whereas two tandem copies are found in gelsolin and villin, suggesting a duplication event. Interestingly, villin displays an additional specific carboxylterminal domain called the "head-piece," which confers on villin F-actin-bundling activity not observed in the others members of the family. It has been postulated that these proteins may have evolved from a common ancestor, by gene duplication in the case of gelsolin and by gene duplication and subsequent addition of a specific domain in the case of villin (13). Seeking support for these postulated evolutionary events, we have examined the exon/intron distribution in the villin gene with respect to the structural and functional domains of the encoded protein (Fig. 5) and then compared this distribution with that of gelsolin. The duplicated domains of human villin are encoded by eight and seven small exons (exons 1-8 and 10-16) and are separated by exon 9, which encodes a short hinge sequence (Fig. 5A). Exons 17 and 18 encode the second linking region and the amino terminus of the head-piece, covering twothirds of this domain. Exon 19 encodes the carboxyl-terminal
region of the head-piece, which is responsible for F-actin bundling activity and is required for the morphogenetic effect induced by villin in transfected fibroblasts (7), as well as the 3' untranslated region of both villin mRNAs. This overall organization reflects the postulated gene duplication of an ancient precursor gene and the addition of a specific domain giving rise to villin. In the two duplicated domains of villin, however, splice sites are not conserved, resulting in a different exon/intron distribution (Fig. SB). Moreover, introns vary in size between the two domains (Fig. 4) and two gaps of 15 and 40 bp appear in the coding sequence of the second domain. The early duplication event has therefore been followed by extensive divergence of the duplicated domains. This exon redistribution and sequence drift may account for the functional difference observed between these two domains, such as the loss of severing activity in the second duplicated domain (23). We have also compared the genomic organization of human gelsolin (19) with that of human villin. Although these two proteins belong to the same family, extensive differences in gene organization are observed. A single gene, much larger than the villin gene (70 kb) but containing only 14 exons, encodes the two gelsolin protein isoforms, a cytoplasmic isoform and a secreted isoform, which are generated by alternative transcriptional start sites and exon skipping. Exons 1 and 2, located far upstream from exon 3, encode the 5' untranslated region of the mRNA encoding cytoplasmic gelsolin. Exon 3 encodes the signal peptide and specific
Proc. Natl. Acad. Sci. USA 88 (1991)
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amino-terminal extension found in secreted gelsolin. In contrast to the first three exons, which are specific to the gelsolin gene, exons 4-14 encode sequences common to the two gelsolin isoforms and are similar to the villin core. This domain is encoded by 16 exons in the case of villin and only 11 exons in the case of gelsolin, indicating an extensive exon redistribution, despite a controlled sequence divergence, since villin and gelsolin share more than 50% homology in their amino acid sequences. The precise organization of exons 6-14 of the gelsolin gene has not been reported. Thus, it is not possible to conclude whether or not the genomic organization of gelsolin also reflects a gene duplication. In conclusion, the structural divergences between the villin and gelsolin genes exclude a simple evolutionary link. After an early gene duplication, these genes may have followed a complex parallel evolution, with the gain of specific domains, such as that allowing secretion, encoded at the 5' end of the gelsolin gene, and that conferring F-actin-bundling activity, encoded at the 3' end of the villin gene. We thank Dr. H. Lehrach for the gift of the EMBL3 human genomic library and Dr. S. Pellegrini for help in performing Southern blot experiments. We thank Dr. M. Buckingham and Dr. R. Kelly for critically reading the manuscript. This work was supported by grants from the Institut National de la Sante et de la Recherche Medicale (no. 86-7008), the Association pour la Recherche sur le Cancer (no. 6379), the Ligue Nationale Franraise contre le Cancer, the Fondation pour la Recherche Mddicale, and the Association Francaise de Lutte contre la Mucoviscidose. 1. Bretscher, A. & Weber, K. (1979) Proc. Nati. Acad. Sci. USA 76, 2321-2325. 2. Bretscher, A. & Weber, K. (1980) Cell 20, 839-847. 3. Craig, S. W. & Powell, L. D. (1980) Cell 22, 739-746. 4. Mooseker, M. S., Graves, T. A., Wharton, K. A., Falco, N. & Howe, C. L. (1980) J. Cell Biol. 87, 809-822. 5. Glenney, J. R., Jr., & Weber, K. (1981) Proc. Natd. Acad. Sci. USA 78, 2810-2814. 6. Glenney, J. R., Jr., Geisler, N., Kaulfus, P. & Weber, K. (1981) J. Biol. Chem. 256, 8156-8161. 7. Friederich, E., Huet, C., Arpin, M. & Louvard, D. (1989) Cell 59, 461-475. 8. Robine, S., Huet, C., Moll, R., Sahuquillo-Merino, C., Coudrier, E., Zweibaum, A. & Louvard, D. (1985) Proc. NatI. Acad. Sci. USA 82, 8488-8492. 9. Boller, K., Arpin, M., Pringault, E., Mangeat, P. & Reggio, H. (1988) Differentiation 39, 51-57. 10. Maunoury, R., Robine, S., Pringault, E., Huet, C., Guenet, J. L., Gaillard, J. A. & Louvard, D. (1988) EMBO J. 7, 3321-3329. 11. Ezzel, R. M., Chafel, M. M. & Matsudaira, P. T. (1989) Development 106, 407-419. 12. Pringault, E. (1990) Ann. Inst. Pasteur (Paris) 2, 120-126. 13. Arpin, M., Pringault, E., Finidori, J., Garcia, A., Jeltsch, J. M., Vandekerckhove, J. & Louvard, D. (1988) J. Cell Biol. 107, 1759-1766. 14. Pringault, E., Arpin, M., Garcia, A., Finidori, J. & Louvard, D. (1986) EMBO J. 5, 3119-3124. 15. Rousseau-Merck, M. F., Simon-Chazottes, D., Arpin, M., Pringault, E., Louvard, D., Gudnet, J. L. & Berger, R. (1988) Hum. Genet. 78, 130-133. 16. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. 17. Frischauf, A. M., Lehrach, H., Poustka, A. M. & Murray, N. (1983) J. Mol. Biol. 170, 827-842. 18. Sanger, F., Nicklen, S., Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 19. Kwiatkowski, D. J., Mehl, R. & Yin, H. L. (1988) J. Cell Biol. 106, 375-384. 20. Kwiatkowski, D. J., Stossel, T. P., Orkin, S. H., Mole, J. E., Colten, H. R. & Yin, H. L. (1986) Nature (London) 323, 455-458. 21. Ampe, C. & Vandekerckhove, J. (1987) EMBO J. 6, 4149-4157. 22. Andre, E., Lottspeich, F., Schleicher, M. & Noegel, A. (1988) J. Biol. Chem. 263, 722-727. 23. Janmey, P. A. & Matsudaira, P. T. (1988) J. Biol. Chem. 263, 16738-16743.
Cell Biology
Structure of the human villin gene (actin-binding protein/gene evolution/intestinal differentiation)
ERIC PRINGAULT, SYLVIE ROBINE,
AND
DANIEL LOUVARD
Unite de Biologie des Membranes, Centre National de la Recherche Scientifique, Unitt Associde 1149, DIpartement de Biologie Molculaire, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France
Communicated by Frangois Jacob, August 26, 1991 (received for review July 12, 1991)
ABSTRACT We have isolated and characterized the complete human villin gene. The villin gene is located on chromosome 2q35-36 in humans and on chromosome 1 in mice. Villin belongs to a family of calcium-regulated actin-binding proteins that share structural and functional homologies. The villin gene is expressed mainly in cells that develop a brush border, such as mucosal cells of the small and large intestine and epithelial cells of the kidney proximal tubules. Villin gene expression is strictly regulated during adult life and embryonic development in the digestive and urogenital tracts and, thus, may be used as a marker of the digestive and renal cell lineages. The human villin gene has one copy per haploid genome, encompasses about 25 kilobases, and contains 19 exons. Analysis of the structural organization of this gene shows that the two mRNAs that encode villin in humans arise by alternative choice of one of the two polyadenylylation signals located within the last exon. The overall organization of the exons reflects the gene duplication event from which this family of actin-binding proteins originated.
structural and functional organization of the protein sequence. We suggest that the two transcripts observed in humans arise from a common pre-mRNA by the random choice of one of two polyadenylylation signals located in the last exon.
MATERIALS AND METHODS Southern Blots. Human genomic DNA was purified from lymphocytes and digested to completion with various restriction enzymes, subjected to electrophoresis, and blotted onto nitrocellulose membranes as described by Southern (16). Blots were then hybridized with three different villin cDNA probes labeled with [a-32P]dCTP by nick-translation. Genomic Library Screening. A genomic library (a gift of H. Lehrach, European Molecular Biology Laboratory, Heidelberg), constructed in phage A by cloning partially Sau3A1digested human lymphocyte genomic DNA into the BamHI sites of the EMBL3 vector (17) was screened with a mixture of overlapping villin cDNA probes spanning the entire length of the mRNA (3.5 kb). Positive clones were then rescreened separately with each probe. Two overlapping clones were selected for further analysis: EMBL3-85e contains the 5' end of the gene, and EMBL3-30a contains the 3' end. DNA Amplification (PCR). Amplification of fragments of the villin gene was performed using the Perkin-Elmer/Cetus thermal cycler and cloned Taq polymerase (Cetus). The temperature cycle was 920C, 1 min; 550C, 1 min; 720C, 3 min (with an increase in elongation time of 2 sec per cycle). The number of cycles was 30-40 depending of the experiment. DNA Sequencing. Exons and the junctions between exons and intervening sequences were sequenced (18) directly on DNA purified from phage EMBL3-85e or -30a. Specific oligonucleotide primers chosen from the coding sequence of the villin cDNA were used. The complete villin cDNA sequence has already been published (13) and has been deposited in the EMBL/GenBank data base (HSVILLR). S1 Nuclease Mapping. A 380-base single-stranded DNA probe spanning the 5' end of the villin mRNA was generated by synthesizing the noncoding strand of a genomic restriction fragment subcloned in the bacteriophage M13 mpl8 with Klenow enzyme, from an oligonucleotide primer located in the first exon of the gene. This probe was labeled by incorporating [a-32P]dCTP during synthesis and purified by electrophoresis in a 6% polyacrylamide gel containing 7 M urea. An aliquot of the probe (10,000 cpm) was hybridized at 48°C for 16 hr in the presence of 10 ,ug of total RNA from either HeLa cells or HT-29 differentiated cells, in 80% formamide/40 mM Pipes/400 mM NaCl/1 mM EDTA with pH adjusted to 6.4. The heteroduplex hybrids were then digested for 1 hr by S1 nuclease (Pharmacia) at concentrations ranging from 0 to 1000 units/ml in 30 mM NaOAc, pH 4.4/280 mM NaCI/4.5 mM Zn(OAc)2 containing salmon
The intestinal mucosa provides an attractive model with which to study the physiological role and the regulation of expression of tissue-specific genes. Arising from stem cells located in the intestinal crypts, immature precursors of enterocytes migrate toward the tips of villi, gradually acquiring their differentiated phenotype. This crypt/villus spatiotemporal gradient of differentiation allows us to establish correlations between the expression of specific proteins and the morphological maturation of enterocytes. One of these proteins, villin, is of particular interest since (i) it belongs to a family of actin-binding proteins that are modulated by calcium (1-6), (ii) it plays a key role in the assembly of the brush border (7), (iii) it is expressed in immature precursors of enterocytes (8-12), and (iv) it displays a strict tissuespecific distribution (8). We previously isolated and characterized a complete cDNA clone coding for human villin (13). The primary structure of villin displays a large duplicated domain common to other actin-severing proteins and a specific additional domain necessary for bundling. Expression of villin mRNA has been studied in various tissues from different species (14). In humans, there are two villin mRNA transcripts; the characterization of the corresponding cDNAs has shown that these two mRNAs differ by an extension of 800 bases in the 3' noncoding region (13). The villin gene maps to human chromosome 2q35-36 and mouse chromosome 1 and belongs to a cluster of genes conserved between the two species (15). Here we report the cloning and characterization of the complete human villin gene. There is one copy of the villin gene per human haploid genome. This gene encompasses about 25 kilobases (kb) and contains 19 exons. The general distribution of exons and intervening sequences reflects the 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.
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sperm DNA at 50 Ag/ml. Protected fragments were analyzed in an 8% polyacrylamide gel containing 7 M urea.
RESULTS AND DISCUSSION Human Haploid Genome Contains One Copy of the Vilhin Gene. Previous in situ hybridization experiments with a villin probe on human chromosomes have provided evidence for the existence of a single locus for the villin gene, on chromosome 2, sub-band q35-36 (15). As there are two villin mRNAs in humans, we used Southern hybridization to examine the number of villin gene copies present at this locus. Total genomic human DNA was digested with various restriction enzymes (EcoRI, BamHI, Bgl II, or combinations), blotted onto nitrocellulose (16), and hybridized with a probe spanning the 5' end of the villin gene from nucleotide (nt) -300 to nt +80 (Fig. 1). This probe contains no restriction sites for the enzymes used. Autoradiography revealed only one hybridizing restriction fragment for each enzyme tested. The experiment was repeated with two different villin cDNA probes, corresponding either to the amino terminus or to the carboxyl terminus of the molecule, giving similar results (data not shown). These data demonstrate that there is only one copy of the villin gene per human haploid genome and that the two mRNAs are therefore transcribed from the same gene. Isolation of the Human Villin Gene. The previously isolated human villin cDNA (13) was used to screen a bacteriophage library of human genomic DNA in the vector EMBL3 (17). After -4 human genome equivalents were screened with a mixture of overlapping probes covering the entire villin cDNA, 12 positive phages were isolated and purified. These positive phages were subjected to restriction enzyme digestion and Southern blotting using either 5' or 3' cDNA probes (data not shown). Two phages containing overlapping inserts, EMBL3-85e and EMBL3-30a, corresponding to the 5' and the 3' regions of the cDNA, respectively, were analyzed in detail. EMBL3-85e contained an insert of about 15 kb, of which 2 kb was 5' flanking sequence and 13 kb was from the 5' region of the villin gene. EMBL3-30a contained an insert of 19 kb, consisting of 13 kb of the 3' region of the villin gene and about 6 kb of 3' flanking sequence. Structure of the Human VilWin Gene. PCR DNA amplification combined with DNA sequence analysis allowed us to elucidate the exon/intron structure of the villin gene. DNA amplification from genomic clones using primers located in 1
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the cDNA allow one to test for the presence or absence of introns by comparing the length of the amplified fragment with the distance separating the two primers on the cDNA. The difference in size between these fragments should represent the size of the intervening sequence. The exact position of each exon/intron boundary was then determined by sequencing the entire coding sequence and a portion of the flanking introns directly from phage DNA containing the villin gene, using coding oligonucleotides as primers. Exon sequences determined from the villin genomic fragments were consistently identical to the villin cDNA sequence previously obtained from a human colon cell line (13). Fig. 2 summarizes the results obtained using this strategy and shows amplified DNA fragments obtained using primers bordering introns 13 and 14. Genomic DNA was amplified using a 5' coding primer beginning at nt 1604 and a 3' noncoding primer ending at nt 1749 of the villin cDNA. The predicted length of coding sequence between these two primers is 145 bp whereas the size of the corresponding genomic fragment was about 850 bp. The difference of about 700 bp represents the length of intervening sequences present between nt 1604 and 1749 of the cDNA. To determine whether these intervening sequences were distributed in one or more introns, the coding sequence and exon/intron boundaries were elucidated in this region of the genomic clone. We found that the intervening sequence of 700 bp represents a single intron located between nt 1702 and 1703 of the cDNA (numbered as intron 13 once the entire gene structure was determined). When the same 5' primer was used with a different 3' primer ending at nt 1849 on the cDNA (predicting a coding sequence of 245 bp), the size of the amplified genomic fragment was about 950 bp, confirming that the length of intervening sequence in this region is about 700 bp and indicating that there are no introns between nt 1749 and 1849. Another example is shown in lane 3, where predicted length of the coding sequence is 200 bp whereas the primers used amplified a genomic fragment of about 1600 bp, suggesting an intervening sequence of about 1400 bp between nt 1794 and nt 1994. Sequence analysis of the genomic fragment
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FIG. 1. Hybridization analysis of genomic fragments from the human villin gene. Aliquots of human genomic DNA (15 ,ug) were digested to completion with EcoRI (lane 1), BamHI (lane 2), BgI II (lane 3), EcoRI/BamHI (lane 4), and EcoRI/Bgl II (lane 5). Restriction fragments were fractionated in a 0.8% agarose gel, denatured, transferred onto nitrocellulose, and hybridized with a partial villin cDNA probe (300 bp) labeled with [a-32P]dCTP by nick-translation.
FIG. 2. Mapping of intervening sequences in the human villin gene by genomic DNA amplification (PCR). Villin gene fragments were amplified from 1 ng of EMBL3-30a phage DNA, using several pairs of primers from villin cDNA, by the Taq polymerase chain reaction. Results of only three amplification experiments are shown. A DNA digests (Boehringer Mannheim) were used as molecular size markers. C, no-DNA control. Lanes 1, amplification with 5' primer beginning at nt 1604 and 3' primer ending at nt 1749 of the cDNA; lanes 2, amplification with 5' primer beginning at nt 1604 and 3' primer ending at nt 1849 of the cDNA; lanes 3, amplification with 5' primer beginning at nt 1794 and 3' primer ending at nt 1994 of the mRNA.
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FIG. 3. Exon/intron organization ofthe human villin gene. Exons are represented by solid boxes numbered 1 to 19 from 5' to 3' in the direction of transcription. The connecting line represents intervening sequences. Question marks indicate three intervening sequences for which DNA amplification products of the intron could not be obtained using flanking primers and thus should be longer than 3 kb. The two EMBL3 phages containing villin genomic DNA that were analyzed in detail are represented below the gene.
showed that this intervening sequence represented a single intron located between nt 1848 and nt 1849 of the cDNA (intron 14). Proceeding along the entire villin gene, we mapped all the intervening sequences by this method. No PCR amplification of fragments longer than 2.5 kb was obtained, and therefore the absence of amplification products suggested the presence of introns of 3 kb or larger. The precise size of these huge introns could not be determined by this method and was not investigated further. Fig. 3 shows the general exon/intron organization of the human villin gene determined by detailed analysis of the two genomic fragments contained in phage (EMBL3-85e and EMBL3-30a). The villin gene encompasses 25 kb and is interrupted by 18 introns ranging in size from 83 bp to 3 kb or larger, such as introns 6, 8, and 9, which yielded no amplified fragments in PCR using flanking primers. Thus the human villin gene contains a total of 19 exons including 18 short exons of 79-203 bp and one larger exon (no. 19) of 1041 bp (Table 1). Sequences of the exon/intron boundaries are shown in Table 2. In all cases, the first two bases of the introns are GT, and the last two bases are AG, in agreement with the consensus sequence of donor and acceptor splice sites. Transcription Initiation Site. To map the site of initiation of villin transcription, an antisense genomic probe overlapping the ATG translation start site of the villin gene was hybridized with total RNA from the intestinal cell line CaCO2 and the heteroduplex was digested with S1 nuclease. Protected fragments were then analyzed in an acrylamide gel (data not shown). Control for the specificity of protection was carried out with total RNA isolated from HeLa cells, which do not Table 1. Exon positions and sizes Exon Position in mRNA, nt*
Size, bp
1-97 97 75 98-172 197 173-369 370-478 109 S 111 479-589 203 6 590-792 7 793-871 79 8 872-970 99 971-1124 154 9 1125-1225 101 10 11 1226-1363 138 12 1364-1522 159 13 1523-1702 180 14 1703-1848 146 15 145 1849-1993 16 1994-2182 189 17 2183-2251 69 141 18 2252-2392 19 2393-3434 1041 *Numbered 5' to 3' in the direction of transcription. 1
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produce villin mRNA. A single fragment was protected from digestion by total RNA isolated from Caco-2 cells but not by total RNA from HeLa cells. The length of the protected fragment was determined by comparison with a sequence ladder from the genomic fragment, obtained using the primer used to synthesize the probe. This allowed us to determine directly the position of the first protected nucleotide. The single product extended 20 nt upstream of the translation initiation site, to the bold underlined cytosine residue of the sequence CCTTCTCCCCCAGGCTCACTCACCATGACC. Overexposure of the gel did not reveal any additional protected fragments (data not shown). This experiment cannot exclude the possibility that an intron longer than 300 bp located just upstream from this cytosine residue could be responsible for the length of the observed protected fragment. Primer extension experiments were unsuccessful, possibly due to the presence of secondary structure on the 5' end of the mRNA. This difficulty was previously encountered when cloning the 5' end of the villin cDNA (13) and has also been reported in the case of the gelsolin gene (19). Nevertheless, the presence of the translation start site at this position can be argued to be due to the absence of an AG splice acceptor site just upstream of the cytosine residue, a feature that appears to be the rule in all introns of the villin gene. Moreover, we have isolated and characterized 2 kb of5' flanking sequence containing putative Table 2. Intron sizes and exon/intron boundaries 3' acceptor 5' donor Intron Size 1 450 ATC GAG gtga ..... ttag GCC ATG 2 1400 CTG GCT gtga ..... ctag ATC CAC 3 700 CTT GT gtag ..... tcag G ATC 4 150 GGA GAG gtag ..... ctag GTA GAG 5 CTC AGG gtaa ..... gcag GGC ATG 1000 6 TAC CA gtga .... . acag T GTG >3000? 7 150 CAC GAG gtaa ..... ctag GAC TGT 8 >3000? GCG CTG gtgt ..... ccag AAC TTC 9 1000 GTG G gtga ..... ctag CC AAA 10 83 GTG CAG gtat ..... ctag GTG TGG 11 600 TGG CAG gtca ..... ccag GGC AGC 12 TAC CAG gtgt ..... gcag GGG GGC 1750 13 700 GGG AAG gtgt ..... ctag GGT TGT 14 1400 AAG AG gtaa ..... gcag A CTA 15 GAC CAG gtag ..... atag GTC TTC 500 16 1500 TGG AGT gtga ..... gcag AAC ACC 17 >3000? ACT GCT gtga ..... ctag GAG GTC 18 900 AAG GAG gtag ..... ctag GAG CAC Introns are numbered from 5' to 3' in the direction of transcription. Approximative sizes of introns determined by DNA amplification are shown. Question marks show three intervening sequence sizes for which DNA amplification products of the intron were never obtained using flanking primers, indicating that the intron is longer than 3 kb (see Results). Splice 5' donor and 3' acceptor sites are also shown. Exon sequences are in uppercase letters. Intron sequences are in lowercase letters. Nucleotides corresponding to the splicing consensus site are in bold letters.
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FIG. 4. Transcription of the two mRNAs from the human villin gene. (Top) Structural organization of villin, represented from the amino terminus to the carboxyl terminus of the protein, showing the large repeated domain (open boxes), homologous to other severing proteins and containing repeated elements (dashed and black boxes), and the specific domain (HP) (see ref. 13). (Middle) The two mRNAs are depicted by horizontal lines. Positions of translation initiation codons (ATG), stop codons (TGA), polyadenylylation signals (AATAA or AATAAA), and poly(A) extensions are indicated. (Bottom) Schematic representation of the structure of the human villin gene. Exons are represented by solid boxes numbered 1 to 19 from 5' to 3' in the direction of transcription. The thin lines connecting one exon to the next represent intervening sequences drawn to scale. Question marks illustrate intervening sequences greater than 3 kb.
upstream transcription factor-responsive elements that promote in vitro transcription of a reporter gene when transfected into cells expressing villin, but not when transfected into villin-negative cells (unpublished observations). Two mRNAs from the Villin Gene. Northern blot hybridization studies have revealed the presence of two mRNA transcripts (2.7 and 3.5 kb) encoding villin in humans (14). By cloning and sequencing the corresponding cDNAs, we have demonstrated that this size difference is entirely due to an
800-nt extension of the 3' noncoding region in the larger mRNA and that coding sequences are identical in the two mRNAs (13). Thus, it is likely that the two mRNAs do not encode different villin protein isoforms. The villin gene appears to have a single transcription start site located 21 nt upstream from the ATG initiation codon, suggesting that these two mRNAs arise from a single precursor. The 3' noncoding region of the larger mRNA must contain the two polyadenylylation signals used to generate the two mRNAs.
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FIG. 5. Intron/exon distribution of the human villin gene compared to structural and functional domains of the protein. (A) Schematic diagrams of the primary structure of villin (U~pper) and of the exon distribution in the human gene (Lower) are aligned. In the protein, large open boxes represent the duplicated domains containing internal repeats (hatched and black boxes). The specific carboxyl-terminal domain is represented by a stippled box (HP). Coding exons are represented by light or dark stippled boxes, whereas 3' noncoding sequence in the last exon is shown by an open box. (B) Exon distributions in the two duplicated domains of the villin gene are aligned.
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Here we demonstrate that the two polyadenylylation signals are located in the last exon (no. 19) of the villin gene (Fig. 4). The two mRNAs differing in size in the 3' noncoding region may therefore be generated by alternative choice of polyadenylylation signal rather than by alternative splicing of exons. Finally, since the two villin mRNAs are expressed in equimolar ratio in all villin-positive tissues (normal and tumor) so far analyzed, and since preferential expression of one transcript has not been observed with respect to the stage of differentiation of intestinal cells or in any cells able to produce villin (14), it is legitimate to suggest that the site of polyadenylylation is randomly selected. The physiological significance of an alternative 3' noncoding extension of villin mRNA is not known. Evolution of the Villin Gene. Villin belongs to a family of four known proteins that share F-actin-severing activity and amino acid sequence homologies (13). These are fragmin and severin, isolated from lower eukaryotes, and gelsolin and villin, found in higher eukaryotes (20-22). These proteins share a large structural domain containing highly conserved repeated motifs, and all four proteins display an F-actinsevering activity. There is one copy of this structural domain in fragmin and severin whereas two tandem copies are found in gelsolin and villin, suggesting a duplication event. Interestingly, villin displays an additional specific carboxylterminal domain called the "head-piece," which confers on villin F-actin-bundling activity not observed in the others members of the family. It has been postulated that these proteins may have evolved from a common ancestor, by gene duplication in the case of gelsolin and by gene duplication and subsequent addition of a specific domain in the case of villin (13). Seeking support for these postulated evolutionary events, we have examined the exon/intron distribution in the villin gene with respect to the structural and functional domains of the encoded protein (Fig. 5) and then compared this distribution with that of gelsolin. The duplicated domains of human villin are encoded by eight and seven small exons (exons 1-8 and 10-16) and are separated by exon 9, which encodes a short hinge sequence (Fig. 5A). Exons 17 and 18 encode the second linking region and the amino terminus of the head-piece, covering twothirds of this domain. Exon 19 encodes the carboxyl-terminal
region of the head-piece, which is responsible for F-actin bundling activity and is required for the morphogenetic effect induced by villin in transfected fibroblasts (7), as well as the 3' untranslated region of both villin mRNAs. This overall organization reflects the postulated gene duplication of an ancient precursor gene and the addition of a specific domain giving rise to villin. In the two duplicated domains of villin, however, splice sites are not conserved, resulting in a different exon/intron distribution (Fig. SB). Moreover, introns vary in size between the two domains (Fig. 4) and two gaps of 15 and 40 bp appear in the coding sequence of the second domain. The early duplication event has therefore been followed by extensive divergence of the duplicated domains. This exon redistribution and sequence drift may account for the functional difference observed between these two domains, such as the loss of severing activity in the second duplicated domain (23). We have also compared the genomic organization of human gelsolin (19) with that of human villin. Although these two proteins belong to the same family, extensive differences in gene organization are observed. A single gene, much larger than the villin gene (70 kb) but containing only 14 exons, encodes the two gelsolin protein isoforms, a cytoplasmic isoform and a secreted isoform, which are generated by alternative transcriptional start sites and exon skipping. Exons 1 and 2, located far upstream from exon 3, encode the 5' untranslated region of the mRNA encoding cytoplasmic gelsolin. Exon 3 encodes the signal peptide and specific
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amino-terminal extension found in secreted gelsolin. In contrast to the first three exons, which are specific to the gelsolin gene, exons 4-14 encode sequences common to the two gelsolin isoforms and are similar to the villin core. This domain is encoded by 16 exons in the case of villin and only 11 exons in the case of gelsolin, indicating an extensive exon redistribution, despite a controlled sequence divergence, since villin and gelsolin share more than 50% homology in their amino acid sequences. The precise organization of exons 6-14 of the gelsolin gene has not been reported. Thus, it is not possible to conclude whether or not the genomic organization of gelsolin also reflects a gene duplication. In conclusion, the structural divergences between the villin and gelsolin genes exclude a simple evolutionary link. After an early gene duplication, these genes may have followed a complex parallel evolution, with the gain of specific domains, such as that allowing secretion, encoded at the 5' end of the gelsolin gene, and that conferring F-actin-bundling activity, encoded at the 3' end of the villin gene. We thank Dr. H. Lehrach for the gift of the EMBL3 human genomic library and Dr. S. Pellegrini for help in performing Southern blot experiments. We thank Dr. M. Buckingham and Dr. R. Kelly for critically reading the manuscript. This work was supported by grants from the Institut National de la Sante et de la Recherche Medicale (no. 86-7008), the Association pour la Recherche sur le Cancer (no. 6379), the Ligue Nationale Franraise contre le Cancer, the Fondation pour la Recherche Mddicale, and the Association Francaise de Lutte contre la Mucoviscidose. 1. Bretscher, A. & Weber, K. (1979) Proc. Nati. Acad. Sci. USA 76, 2321-2325. 2. Bretscher, A. & Weber, K. (1980) Cell 20, 839-847. 3. Craig, S. W. & Powell, L. D. (1980) Cell 22, 739-746. 4. Mooseker, M. S., Graves, T. A., Wharton, K. A., Falco, N. & Howe, C. L. (1980) J. Cell Biol. 87, 809-822. 5. Glenney, J. R., Jr., & Weber, K. (1981) Proc. Natd. Acad. Sci. USA 78, 2810-2814. 6. Glenney, J. R., Jr., Geisler, N., Kaulfus, P. & Weber, K. (1981) J. Biol. Chem. 256, 8156-8161. 7. Friederich, E., Huet, C., Arpin, M. & Louvard, D. (1989) Cell 59, 461-475. 8. Robine, S., Huet, C., Moll, R., Sahuquillo-Merino, C., Coudrier, E., Zweibaum, A. & Louvard, D. (1985) Proc. NatI. Acad. Sci. USA 82, 8488-8492. 9. Boller, K., Arpin, M., Pringault, E., Mangeat, P. & Reggio, H. (1988) Differentiation 39, 51-57. 10. Maunoury, R., Robine, S., Pringault, E., Huet, C., Guenet, J. L., Gaillard, J. A. & Louvard, D. (1988) EMBO J. 7, 3321-3329. 11. Ezzel, R. M., Chafel, M. M. & Matsudaira, P. T. (1989) Development 106, 407-419. 12. Pringault, E. (1990) Ann. Inst. Pasteur (Paris) 2, 120-126. 13. Arpin, M., Pringault, E., Finidori, J., Garcia, A., Jeltsch, J. M., Vandekerckhove, J. & Louvard, D. (1988) J. Cell Biol. 107, 1759-1766. 14. Pringault, E., Arpin, M., Garcia, A., Finidori, J. & Louvard, D. (1986) EMBO J. 5, 3119-3124. 15. Rousseau-Merck, M. F., Simon-Chazottes, D., Arpin, M., Pringault, E., Louvard, D., Gudnet, J. L. & Berger, R. (1988) Hum. Genet. 78, 130-133. 16. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. 17. Frischauf, A. M., Lehrach, H., Poustka, A. M. & Murray, N. (1983) J. Mol. Biol. 170, 827-842. 18. Sanger, F., Nicklen, S., Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 19. Kwiatkowski, D. J., Mehl, R. & Yin, H. L. (1988) J. Cell Biol. 106, 375-384. 20. Kwiatkowski, D. J., Stossel, T. P., Orkin, S. H., Mole, J. E., Colten, H. R. & Yin, H. L. (1986) Nature (London) 323, 455-458. 21. Ampe, C. & Vandekerckhove, J. (1987) EMBO J. 6, 4149-4157. 22. Andre, E., Lottspeich, F., Schleicher, M. & Noegel, A. (1988) J. Biol. Chem. 263, 722-727. 23. Janmey, P. A. & Matsudaira, P. T. (1988) J. Biol. Chem. 263, 16738-16743.
