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A Human Protective Protein Gene Partially Overlaps the Gene Encoding Phospholipid Transfer Protein on the Complementary Strand of DNA
JOBNAME: BBRC 220#3 PAGE: 1 SESS: 2 OUTPUT: Sun May 5 12:46:08 1996 /xypage/worksmart/tsp000/69829b/66
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
220, 802–806 (1996)
0484
A Human Protective Protein Gene Partially Overlaps the Gene Encoding Phospholipid Transfer Protein on the Complementary Strand of DNA Michie Shimmoto,*,1 Yutaka Nakahori,† Ikumi Matsushita,† Toshikatsu Shinka,†,‡ Yoko Kuroki,*,† Kohji Itoh,* and Hitoshi Sakuraba* *Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan; †Department of Human Genetics, School of International Health, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan; and ‡Department of Pediatrics, Tohoku University School of Medicine, 2-1 Seiryocho, Sendai 980-77, Japan Received February 16, 1996 The entire human protective protein gene has been cloned, and structural analysis revealed that the gene spans 7.5kb and comprises 15 exons. Furthermore, it partially overlaps on the opposite strand with the gene encoding phospholipid transfer protein. This region of DNA on chromosome 20 appears to encode two distinct mRNAs expressing defined functional products, and the mRNAs overlap by 58 nucleotides at their 39-untranslated ends. © 1996 Academic Press, Inc.
Protective protein is a multifunctional glycoprotein which regulates the expression of b-galactosidase and neuraminidase, by stabilizing the former and by activating the latter, through the formation of multienzymic complex in lysosomes (1–3). Furthermore, protective protein exhibits catalytic activity, acting as a carboxypeptidase at acidic pH, and as an esterase and a carboxylterminal deamidase at neutral pH (4). A genetic defect of protective protein causes a lysosomal disorder, galactosialidosis, associated with a combined deficiency of carboxypeptidase/esterase/ deamidase, b-galactosidase and neuraminidase (5–7). Patients with galactosialidosis exhibit systemic manifestations (8,9). Most of them develop ocular and neurological deterioration, dysmorphism and angiokeratoma. Severe cases develop visceromegaly, and renal and cardiac involvement. Galactosialidosis is transmitted as an autosomal recessive trait, and the human gene encoding protective protein has been localized on chromosome 20q13.1 (10). A cDNA encoding human protective protein was cloned by Galjart et al (11). It directs the synthesis of a 452 amino acid precursor molecule and recognizes a 2-kb mRNA in fibroblasts. But the genomic organization has not been fully elucidated yet. Phospholipid transfer protein (PLTP) promotes the exchange and transfer of phospholipids (12). PLTP also has the ability to facilitate modulation of high density lipoprotein size and composition (13), and is expected to protect against coronary heart disease. The entire human PLTP cDNA is 1,750 base pairs in length, and contains an open reading frame of 1,518 nucleotides (nt) encoding a leader of 17 amino acids and a mature protein of 476 residues (14). Recently, the PLTP gene was mapped to chromosome 20q12-q13.1 and its structural organization has been roughly determined (15,16). In this study, we determined the entire structure of the human protective protein gene, and demonstrate that protective protein mRNA and PLTP mRNA are encoded by two partially overlapped genes on the complementary strand of DNA. MATERIALS AND METHODS Isolation of clones for the protective protein gene. The YAC library of human DNA from the Centre d’Etude du Polymaophisme Humain (CEPH YAC library) was screened by means of PCR-base danalysis (17) using a set of oligo1
To whom correspondence should be addressed. Fax: 81-3-3823-6008. 802
0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
nucleotide primers complementary to the sequences of DNA in exons 3 and 6 of the protective protein gene. A positive clone, Y640 (640B9) was isolated and high molecular weight YAC-containing yeast DNA from the clone was prepared by the standard method. Subsequently, a cosmid sublibrary was constructed from the YAC DNA using a cosmid vector, SuperCos1 (Stratagene), according to the manufacturer’s method. Then colony hybridization using 32P-labelled protective protein cDNA as a probe was performed and 35 positive cosmid clones were obtained from the sublibrary. Cosmid DNA was prepared from each clone, digested with HindIII and then subjected to Southern blotting. A cosmid clone, Cos36, which contained 3 positive HindIII DNA fragments (sizes; 4.3, 1.6 and 9.5kb) was isolated. The three HindIII DNA fragments were purified and subcloned into a plasmid vector pUC18 (Nippon Gene) and resulting clones were named as Y640H4.3pU, Y640H1.6pU and Y640H9.5pU, respectively (Fig. 1). PCR and restriction analysis. To determine the organizations of the protective protein and PLTP genes, PCR was performed using these clones and genomic DNA from human subjects as samples. The PCR products are shown in Fig. 1. Restriction digestion of the fragment, PPg7, amplified from the above clones and human genomic DNA, was performed with Pvu II, Pst I, Taq I and Msp I. DNA sequencing and homology search. The plasmids, Y640H4.3pU, Y640H1.6pU and Y640H9.5pU, and deleted plasmids of Y640H9.5pU prepared using a deletion kit (Nippon Gene) were sequenced using a DyDeoxy Terminator Cycle Sequencing kit (Applied Biosystems) and an automated DNA sequencer (Applied Biosystems; Model 373A). PCRamplified DNA fragments were directly sequenced. A homology search was performed using the GenomNet WWW service and GenBank (Machine address; http:// www.genome.ad.jp/).
RESULTS AND DISCUSSION We screened a yeast artificial chromosome (YAC) library by means of PCR-based analysis, and obtained a clone, Y640, covering the protective protein gene. A cosmid sublibrary was constructed from Y640 and screened by colony hybridization. The 35 positive clones among the 6 × 103 clones were isolated from the sublibrary. A cosmid clone containing three HindIII DNA fragments (Y640H4.3, Y640H1.6 and Y640H9.5) was selected by Southern blotting using the protective protein cDNA as a probe (Fig. 1). These HindIII DNA fragments were inserted into plasmid vector pUC18, and the resulting clones were designated as Y640H4.3pU, Y640H1.6pU and Y640H9.5pU, respectively. Sequencing analysis revealed that clones Y640H4.3pU and Y640H1.6pU had identical nucleotide sequences to those of chr20-4 and chr20-5, respectively. Chr20-4 and chr20-5 were derived from a human chromosome 20 specific library, and cover the 59-end and central region of the protective protein gene, respectively, as described previously (18). Y640H9.5pU contains the nucleotide sequence of the remainder of the 39-region of the gene. A diagram of the organization of the protective protein gene is shown in Fig. 1. The gene, which spans 7.5kb, comprises 15 exons. The location and size of each exon and intron, as well as the nucleotide sequences at the intron/exon junctions, are presented in Table 1. All intron/exon junctions satisfied the GT/AG rule (19). The first exon contains the 59-untranslated region and exons 2–15 encode the precursor of protective protein. The last exon contains the 39-untranslated region and polyadenylation signal. A homology search revealed that the sequence of the 39-end of the protective protein gene was identical with that of PLTP cDNA in the reverse direction, and 58nt of the 39-untranslated regions of these cDNAs were complementary. To examine the localization of both genes, PCR was performed using DNAs from Y640H9.5pU and Y640 as templates. Fragment PLg1 was amplified from both clones, and fragments PLg2, PLg3 and PLg4 from Y640 (Fig. 1). Direct sequencing analysis demonstrated that these fragments covered the entire PLTP gene. PLg 1 contained a part of exon 11 and exons 12–16 of the PLTP gene, and a part of exon 15 of the protective protein gene, and the 39-ends of these genes overlapped. Furthermore, amplification of genomic DNA from unrelated human subjects followed by restriction digestion confirmed this result. The overlapped sequence does not contain an open reading frame, but it does contain the polyadenylation signal of protective protein (Fig. 1). This region is thought to be necessary for the transcription of protective protein. To date, only a few overlapping genes have been reported in mammalian cells (20–25), especially ones expressing defined functional products. In our case, it is not clear whether or not the partially overlapping genes are involved in mutual gene regulation. The protective protein gene is one of the 803
Vol. 220, No. 3, 1996
FIG. 1. Organization of the human protective protein gene and the phospholipid transfer protein gene. Exons are represented by boxes and numbered. The solid boxes denote the 59- and 39-untranslated regions. Vertical lines indicate the Hind III restriction sites. The inserted fragments in pUCl8 clones and PCR fragments obtained to complete the analysis of the entire gene are also shown. The overlapped region in cDNA is boxed in the center of the figure. A putative polyadenylation signal is indicated by an underline.
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804
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1 Exon–Intron Organization of the Protective Protein Gene
Exon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 a b
Exon size (bp)
Location of exona
$27 194 112 51 87 156 91 86 91 79 141 76 90 105 450b
– −1 1–194 195–306 307–357 358–444 445–600 601–691 692–777 778–868 869–947 948–1088 1089–1164 1165–1254 1255–1359 1360–1809
39 Splice site (acceptor)
59 Splice site (donor)
------GCACAGgtgagctggcaccgg-----tgctgcctcccgtagATGATC------CTACTGgtctgccgccctgcc-----tgacccccctcccagGTTTGT------TTCCTGgtgagtggacagcag-----ctctttcctcctcagGTCCAG------AATCTGgtatagctggagctg-----ttccctcccctctagATTGCC------ACTGAGgtgagtctggtgcct-----ctctgccatccccagGTCGCC------CTTCAGgtgcagggtagctgc-----ctgggtgtttcacagGGGCTG------GAACAGgtatgggatagggca-----tctcatctcctacagGCTTTG------ACCAATgtgaggttctgccat-----tgttgtatcattcagCTTCAG------TTTTAGgtaggtgctgctggg-----cacctcacattgcagGTATGA------CATCAGgtgtgcaagggcgtg-----gtctgtgccttccagGCACTG------GTGCAAgtgaggttctgtggc-----gtatgttcccggcagCTTTCT------TCACAGgtgagtggggagagc-----cccatcctgctttagAAATAC------CAGAAGgtaaggtagagattc-----ttcctggtggggcagATGGAG------ATCAAGgtagggactgggcct-----aacattgctcctcagGGCGCC------AGCTTC
Intron (kb) 0.19 0.15 0.24 0.07 0.24 0.34 0.67 0.58 0.08 0.09 1.84 0.67 0.19 0.26
Counting from A of the initiation codon in cDNA. To the polyadenylation signal.
house-keeping genes and is believed to express the RNA transcript in almost all tissues (26). PLTP is also expressed in many tissues (14). So it seems that these partially overlapping transcripts are expressed in the same tissue without suppression. We confirmed that the 39-end of the mouse protective protein gene is located very close to that of the PLTP gene on the opposite strand of DNA (data not shown), and the possibility of gene overlapping is now under study. Thus, evidence that the genome of mammalian cells encodes two distinct genes which express functional products by using both strands on the same DNA has been obtained. ACKNOWLEDGMENTS This work was supported by grants from the Ministry of Education, Science and Culture of Japan, the Ministry of Health and Welfare of Japan, the Naito Foundation, and the Ono Medical Research Foundation.
REFERENCES 1. Hoogeveen, A. T., Verheijen, F. W., and Galjaard, H. (1983) J. Biol. Chem. 258, 12143–12146. 2. Verheijen, F. W., Palmeri, S., Hoogeveen, A. T., and Galjaard, H. (1985) Eur. J. Biochem. 149, 315–321. 3. d’Azzo, A., Hoogeveen, A. T., Reuser, A. J. J., Robinson, D., and Galjaard, H. (1982) Proc. Natl. Acad. Sci. USA 79, 4535–4539. 4. Itoh, K., Takiyama, N., Kase, R., Kondoh, K., Sano, A., Oshima, A., Sakuraba, H., and Suzuki, Y. (1993) J. Biol. Chem. 268, 1180–1186. 5. Tranchemontagne, J., Michaud, L., and Potier, M. (1990) Biochem. Biophys. Res. Commun. 168, 22–29. 6. Kase, R., Itoh, K., Takiyama, N., Oshima, A., Sakuraba, H., and Suzuki, Y. (1990) Biochem. Biophys. Res. Commun. 172, 1175–1179. 7. Itoh, K., Takiyama, N., Nagao, Y., Oshima, A., Sakuraba, H., and Suzuki, Y. (1991) Jpn. J. Hum. Genet. 36, 171–177. 8. d’Azzo, A., Andria, G., Strisciuglio, P., and Galjaard, H. (1995) in The Metabolic and Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, C. R., Sly, W. S., and Valle, D., Eds.), 7th ed., pp. 2825–2835, McGraw–Hill, New York. 9. Takano, T., Shimmoto, M., Fukuhara, Y., Itoh, K., Kase, R., Takiyama, N., Kobayashi, T., Oshima, A., Sakuraba, H., and Suzuki, Y. (1992) Brain Dysfunct. 4, 271–280. 10. Wiegant, J., Galjart, N. J., Raap, A. K., and d’Azzo, A. (1991) Genomics 10, 345–349. 11. Galjart, N. J., Gillemans, N., Harris, A., van der Horst, G. T. J., Verheijen, F. W., Galjaard, H., and d’Azzo, A. (1988) Cell 54, 755–764. 805
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12. Tollefson, J. H., Ravik, S., and Albers, J. J. (1988) J. Lipid Res. 29, 1593–1602. 13. Jauhiainen, M., Metso, J., Pahlman, R., Blamqvist, S., Tol, A., and Ehnholm, C. (1988) J. Biol. Chem. 268, 4032–4036. 14. Day, J. R., Albers, J. J., Lofton-Day, C. E., Ching, A. F. A., Grant, F. J., O’Hara, P. J., Marcovina, S. M., and Adolphson, J. L. (1994) J. Biol. Chem. 269, 9388–9391. 15. Whitmore, T. E., Day, J. R., and Albers, J. J. (1995) Genomics 28, 599–600. 16. Tu, A.-Y., Deeb, S. S., Iwasaki, L., Day, J. R., and Albers, J. J. (1995) Biochem. Biophys. Res. Commun. 207, 552–558. 17. Green, E. D., and Olson, M. V. (1990) Proc. Natl. Acad. Sci. USA 87, 1213–1217. 18. Shimmoto, M., Takano, T., Fukuhara, Y., Oshima, A., Sakuraba, H., and Suzuki, Y. (1990) Proc. Jpn. Acad. 66B, 217–222. 19. Mount, S. M. (1982). A catalogue of splice junction sequences. Nucleic Acids Res. 10, 459–472. 20. Williams, T., and Fried, M. (1986) Nature 322, 275–279. 21. Adelman, J. P., Bord, C. T., Dougolass, J., and Herbert, E. (1987) Science 235, 1514–1517. 22. Lazar, M. A., Hodin, R. A., Darling, D. S., and Chin, W. W. (1989) Mol. Cell. Biol. 9, 1128–1136. 23. Miyajima, N., Horiuchi, R., Shibuya, Y., Fukushige, S., Matsubara, K., Toyoshima, K., and Yamamoto, T. (1989) Cell 57, 31–39. 24. Shayiq, R. M., and Avadhani, N. G. (1992) J. Biol. Chem. 267, 2421–2428. 25. Bristow, J., Tee, M. K., Gitelman, S. E., Mellon, S., and Miller, W. L. (1993) J. Cell. Biol. 122, 265–278. 26. Satake, A., Itoh, K., Shimmoto, M., Saido, T. C., Sakuraba, H., and Suzuki, Y. (1994) Biochem. Biophys. Res. Commun. 205, 38–43.
806
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
220, 802–806 (1996)
0484
A Human Protective Protein Gene Partially Overlaps the Gene Encoding Phospholipid Transfer Protein on the Complementary Strand of DNA Michie Shimmoto,*,1 Yutaka Nakahori,† Ikumi Matsushita,† Toshikatsu Shinka,†,‡ Yoko Kuroki,*,† Kohji Itoh,* and Hitoshi Sakuraba* *Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan; †Department of Human Genetics, School of International Health, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan; and ‡Department of Pediatrics, Tohoku University School of Medicine, 2-1 Seiryocho, Sendai 980-77, Japan Received February 16, 1996 The entire human protective protein gene has been cloned, and structural analysis revealed that the gene spans 7.5kb and comprises 15 exons. Furthermore, it partially overlaps on the opposite strand with the gene encoding phospholipid transfer protein. This region of DNA on chromosome 20 appears to encode two distinct mRNAs expressing defined functional products, and the mRNAs overlap by 58 nucleotides at their 39-untranslated ends. © 1996 Academic Press, Inc.
Protective protein is a multifunctional glycoprotein which regulates the expression of b-galactosidase and neuraminidase, by stabilizing the former and by activating the latter, through the formation of multienzymic complex in lysosomes (1–3). Furthermore, protective protein exhibits catalytic activity, acting as a carboxypeptidase at acidic pH, and as an esterase and a carboxylterminal deamidase at neutral pH (4). A genetic defect of protective protein causes a lysosomal disorder, galactosialidosis, associated with a combined deficiency of carboxypeptidase/esterase/ deamidase, b-galactosidase and neuraminidase (5–7). Patients with galactosialidosis exhibit systemic manifestations (8,9). Most of them develop ocular and neurological deterioration, dysmorphism and angiokeratoma. Severe cases develop visceromegaly, and renal and cardiac involvement. Galactosialidosis is transmitted as an autosomal recessive trait, and the human gene encoding protective protein has been localized on chromosome 20q13.1 (10). A cDNA encoding human protective protein was cloned by Galjart et al (11). It directs the synthesis of a 452 amino acid precursor molecule and recognizes a 2-kb mRNA in fibroblasts. But the genomic organization has not been fully elucidated yet. Phospholipid transfer protein (PLTP) promotes the exchange and transfer of phospholipids (12). PLTP also has the ability to facilitate modulation of high density lipoprotein size and composition (13), and is expected to protect against coronary heart disease. The entire human PLTP cDNA is 1,750 base pairs in length, and contains an open reading frame of 1,518 nucleotides (nt) encoding a leader of 17 amino acids and a mature protein of 476 residues (14). Recently, the PLTP gene was mapped to chromosome 20q12-q13.1 and its structural organization has been roughly determined (15,16). In this study, we determined the entire structure of the human protective protein gene, and demonstrate that protective protein mRNA and PLTP mRNA are encoded by two partially overlapped genes on the complementary strand of DNA. MATERIALS AND METHODS Isolation of clones for the protective protein gene. The YAC library of human DNA from the Centre d’Etude du Polymaophisme Humain (CEPH YAC library) was screened by means of PCR-base danalysis (17) using a set of oligo1
To whom correspondence should be addressed. Fax: 81-3-3823-6008. 802
0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
nucleotide primers complementary to the sequences of DNA in exons 3 and 6 of the protective protein gene. A positive clone, Y640 (640B9) was isolated and high molecular weight YAC-containing yeast DNA from the clone was prepared by the standard method. Subsequently, a cosmid sublibrary was constructed from the YAC DNA using a cosmid vector, SuperCos1 (Stratagene), according to the manufacturer’s method. Then colony hybridization using 32P-labelled protective protein cDNA as a probe was performed and 35 positive cosmid clones were obtained from the sublibrary. Cosmid DNA was prepared from each clone, digested with HindIII and then subjected to Southern blotting. A cosmid clone, Cos36, which contained 3 positive HindIII DNA fragments (sizes; 4.3, 1.6 and 9.5kb) was isolated. The three HindIII DNA fragments were purified and subcloned into a plasmid vector pUC18 (Nippon Gene) and resulting clones were named as Y640H4.3pU, Y640H1.6pU and Y640H9.5pU, respectively (Fig. 1). PCR and restriction analysis. To determine the organizations of the protective protein and PLTP genes, PCR was performed using these clones and genomic DNA from human subjects as samples. The PCR products are shown in Fig. 1. Restriction digestion of the fragment, PPg7, amplified from the above clones and human genomic DNA, was performed with Pvu II, Pst I, Taq I and Msp I. DNA sequencing and homology search. The plasmids, Y640H4.3pU, Y640H1.6pU and Y640H9.5pU, and deleted plasmids of Y640H9.5pU prepared using a deletion kit (Nippon Gene) were sequenced using a DyDeoxy Terminator Cycle Sequencing kit (Applied Biosystems) and an automated DNA sequencer (Applied Biosystems; Model 373A). PCRamplified DNA fragments were directly sequenced. A homology search was performed using the GenomNet WWW service and GenBank (Machine address; http:// www.genome.ad.jp/).
RESULTS AND DISCUSSION We screened a yeast artificial chromosome (YAC) library by means of PCR-based analysis, and obtained a clone, Y640, covering the protective protein gene. A cosmid sublibrary was constructed from Y640 and screened by colony hybridization. The 35 positive clones among the 6 × 103 clones were isolated from the sublibrary. A cosmid clone containing three HindIII DNA fragments (Y640H4.3, Y640H1.6 and Y640H9.5) was selected by Southern blotting using the protective protein cDNA as a probe (Fig. 1). These HindIII DNA fragments were inserted into plasmid vector pUC18, and the resulting clones were designated as Y640H4.3pU, Y640H1.6pU and Y640H9.5pU, respectively. Sequencing analysis revealed that clones Y640H4.3pU and Y640H1.6pU had identical nucleotide sequences to those of chr20-4 and chr20-5, respectively. Chr20-4 and chr20-5 were derived from a human chromosome 20 specific library, and cover the 59-end and central region of the protective protein gene, respectively, as described previously (18). Y640H9.5pU contains the nucleotide sequence of the remainder of the 39-region of the gene. A diagram of the organization of the protective protein gene is shown in Fig. 1. The gene, which spans 7.5kb, comprises 15 exons. The location and size of each exon and intron, as well as the nucleotide sequences at the intron/exon junctions, are presented in Table 1. All intron/exon junctions satisfied the GT/AG rule (19). The first exon contains the 59-untranslated region and exons 2–15 encode the precursor of protective protein. The last exon contains the 39-untranslated region and polyadenylation signal. A homology search revealed that the sequence of the 39-end of the protective protein gene was identical with that of PLTP cDNA in the reverse direction, and 58nt of the 39-untranslated regions of these cDNAs were complementary. To examine the localization of both genes, PCR was performed using DNAs from Y640H9.5pU and Y640 as templates. Fragment PLg1 was amplified from both clones, and fragments PLg2, PLg3 and PLg4 from Y640 (Fig. 1). Direct sequencing analysis demonstrated that these fragments covered the entire PLTP gene. PLg 1 contained a part of exon 11 and exons 12–16 of the PLTP gene, and a part of exon 15 of the protective protein gene, and the 39-ends of these genes overlapped. Furthermore, amplification of genomic DNA from unrelated human subjects followed by restriction digestion confirmed this result. The overlapped sequence does not contain an open reading frame, but it does contain the polyadenylation signal of protective protein (Fig. 1). This region is thought to be necessary for the transcription of protective protein. To date, only a few overlapping genes have been reported in mammalian cells (20–25), especially ones expressing defined functional products. In our case, it is not clear whether or not the partially overlapping genes are involved in mutual gene regulation. The protective protein gene is one of the 803
Vol. 220, No. 3, 1996
FIG. 1. Organization of the human protective protein gene and the phospholipid transfer protein gene. Exons are represented by boxes and numbered. The solid boxes denote the 59- and 39-untranslated regions. Vertical lines indicate the Hind III restriction sites. The inserted fragments in pUCl8 clones and PCR fragments obtained to complete the analysis of the entire gene are also shown. The overlapped region in cDNA is boxed in the center of the figure. A putative polyadenylation signal is indicated by an underline.
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804
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1 Exon–Intron Organization of the Protective Protein Gene
Exon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 a b
Exon size (bp)
Location of exona
$27 194 112 51 87 156 91 86 91 79 141 76 90 105 450b
– −1 1–194 195–306 307–357 358–444 445–600 601–691 692–777 778–868 869–947 948–1088 1089–1164 1165–1254 1255–1359 1360–1809
39 Splice site (acceptor)
59 Splice site (donor)
------GCACAGgtgagctggcaccgg-----tgctgcctcccgtagATGATC------CTACTGgtctgccgccctgcc-----tgacccccctcccagGTTTGT------TTCCTGgtgagtggacagcag-----ctctttcctcctcagGTCCAG------AATCTGgtatagctggagctg-----ttccctcccctctagATTGCC------ACTGAGgtgagtctggtgcct-----ctctgccatccccagGTCGCC------CTTCAGgtgcagggtagctgc-----ctgggtgtttcacagGGGCTG------GAACAGgtatgggatagggca-----tctcatctcctacagGCTTTG------ACCAATgtgaggttctgccat-----tgttgtatcattcagCTTCAG------TTTTAGgtaggtgctgctggg-----cacctcacattgcagGTATGA------CATCAGgtgtgcaagggcgtg-----gtctgtgccttccagGCACTG------GTGCAAgtgaggttctgtggc-----gtatgttcccggcagCTTTCT------TCACAGgtgagtggggagagc-----cccatcctgctttagAAATAC------CAGAAGgtaaggtagagattc-----ttcctggtggggcagATGGAG------ATCAAGgtagggactgggcct-----aacattgctcctcagGGCGCC------AGCTTC
Intron (kb) 0.19 0.15 0.24 0.07 0.24 0.34 0.67 0.58 0.08 0.09 1.84 0.67 0.19 0.26
Counting from A of the initiation codon in cDNA. To the polyadenylation signal.
house-keeping genes and is believed to express the RNA transcript in almost all tissues (26). PLTP is also expressed in many tissues (14). So it seems that these partially overlapping transcripts are expressed in the same tissue without suppression. We confirmed that the 39-end of the mouse protective protein gene is located very close to that of the PLTP gene on the opposite strand of DNA (data not shown), and the possibility of gene overlapping is now under study. Thus, evidence that the genome of mammalian cells encodes two distinct genes which express functional products by using both strands on the same DNA has been obtained. ACKNOWLEDGMENTS This work was supported by grants from the Ministry of Education, Science and Culture of Japan, the Ministry of Health and Welfare of Japan, the Naito Foundation, and the Ono Medical Research Foundation.
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