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Cloning, tissue-specific expression, gene structure and chromosomal localization of human phosphatidylcholine transfer protein1
Biochimica et Biophysica Acta 1447 (1999) 265^270 www.elsevier.com/locate/bba
Short sequence-paper
Cloning, tissue-speci¢c expression, gene structure and chromosomal localization of human phosphatidylcholine transfer protein1 David E. Cohen
a;
*, Richard M. Green b , Michele K. Wu a , David R. Beier
c
a
Departments of Medicine and Biochemistry, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Ullmann Building Room 625, 1300 Morris Park Avenue, Bronx, NY 10461, USA b Division of Digestive and Liver Diseases, University of Illinois at Chicago and West Side VAMC, 840 S. Wood St. M/C 787, Chicago, IL 60612, USA c Division of Genetics, Harvard Medical School, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA Received 29 June 1999; accepted 20 August 1999
Abstract Phosphatidylcholine transfer protein (PC-TP) is a cytosolic protein that catalyzes intermembrane transfer of phosphatidylcholines in vitro. We have cloned a cDNA encoding the human ortholog of PC-TP and have determined its tissue-specific expression as well as genomic organization. Radiation hybrid mapping localized the human gene, PCTP, to chromosome 17q21-22 and PCR-based single strand conformation polymorphism analysis of an interspecific backcross assigned mouse Pctp to the region of syntenic conservation on chromosome 11. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Phospholipid; Lipid; Transfer Protein; Liver; Bile
Phosphatidylcholine transfer protein (PC-TP) is a soluble protein that was ¢rst puri¢ed from bovine and rat liver more than two decades ago by virtue of its ability to catalyze intermembrane transfer of phosphatidylcholines exclusive of other phospholipid classes [1]. Although well characterized biochemically, the physiological function(s) of PC-TP remains unknown. Because phospholipids in bile are enriched ( s 95%) in phosphatidylcholines [2] and because PCTP is highly expressed in rat liver [3], we have hypothesized that PC-TP might function in hepatocel* Corresponding author. Fax: +1-718-430-8975; E-mail: [email protected] 1 The nucleotide sequence data in this paper have been submitted to GenBank and have been assigned accession numbers AF114430^AF114437.
lular selection and transport of phosphatidylcholines during bile formation [4]. In support of this possibility, we have shown that bile salts markedly stimulate in vitro activity of puri¢ed bovine liver PC-TP [4], and LaMorte et al. [5] have demonstrated recently that PC-TP displays highest a¤nities for the molecular species of phosphatidylcholines that are secreted into bile. Cloning of full-length or partial cDNAs has demonstrated that PC-TPs from cow [6,7], rat [7,8] and mouse [7] are highly homologous. To facilitate cDNA cloning of human PC-TP, the nucleotide sequence of a cDNA encoding bovine PC-TP [6] was utilized to search the expressed sequence tag (EST) database (http://www.ncbi.nlm.nih.gov/dbEST/) [9]. Clones with 5P and 3P nucleotide sequences similar to bovine PC-TP were identi¢ed (GenBank accession
0167-4781 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 9 ) 0 0 1 6 3 - 3
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Fig. 1. Nucleotide sequence of the 1932 bp cDNA encoding human PC-TP. Dots (...) indicate abbreviation of the 3P-untranslated region (UTR). Underlined nucleotide sequence was obtained from dbEST clones (GenBank accession numbers W02400 and N70264) as described in the text. The remainder of the nucleotide sequence was determined by 5PRACE and has been deposited in GenBank with accession number AF114430. The deduced 214 aa of the ORF are shown with the stop codon indicated by an asterisk (*).
numbers W02400 and N70264, respectively). The former clone was incomplete at its 5P end, and therefore rapid ampli¢cation of the 5P cDNA end (5PRACE) was employed in order to obtain a complete open reading frame (ORF). Because PC-TP mRNA is abundant in both mouse liver and kidney [7], human kidney cDNA (Clontech Marathon-Ready cDNA) was utilized as template for PCR together with gene speci¢c antisense primers (5P-CAAACAGTCTGAGGTATCCCAC-3P and 5P-TGAGGTAGTTCTGACAGGCTCTTGC-3P) designed to the dbEST nucleotide sequences. The nucleotide and deduced amino acid sequences of the composite cDNA encoding human PC-TP is presented in Fig. 1. Human PCTP is a 214 aa (24 842 Da) protein that is 76% and 80% identical to bovine [6,7] and rat [8] PC-TPs, respectively. In order to examine tissue-speci¢c expression, the cDNA from Fig. 1 was utilized to probe human multiple tissue Northern blots (Clontech). Fig. 2 demonstrates that highest PC-TP mRNA levels
were present in liver, placenta, testis, kidney and heart. Only thymus lacked evidence of PC-TP expression and low levels were observed in brain and lung. All other tissues demonstrated appreciable PC-TP mRNA levels. By comparison, Teerlink et al. [3] in an earlier study employed a radioimmunoassay to quantify PC-TP in rat tissues: Highest concentrations were found in liver followed by intestinal mucosa, kidney, spleen, lung and adrenals. In the adult mouse, expression of PC-TP mRNA [7] was highest in kidney, liver and testis, with low levels detected in most tissues and minimal levels in brain and thymus. In order to determine the structure of the human phosphatidylcholine transfer protein gene PCTP, the cDNA cloned by 5PRACE was utilized to screen ¢lters of a gridded RPCI Human PAC Library (RPCI1, Roswell Park Cancer Institute, Department of Human Genetics, Bu¡alo, NY, USA). Of the seven clones that hybridized (26L8, 30Mll, 30Tll, 77D6, 82M5, 108P13 and 296B16), Southern blot analysis of EcoRI digested DNA demonstrated that all but
BBAEXP 91309 19-10-99
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one (108P13) contained PCTP in its entirety. Hybridizing bands from one of the clones (296B16) were subcloned into pBluescript KS (Stratagene) for nucleotide sequence analysis. Utilizing primers designed to the cDNA, nucleotide sequences of exons as well as intron-exon junctions were determined. Intron sizes were determined by nucleotide sequencing and by PCR using exon-speci¢c primers. The structure of PCTP is presented schematically in Fig. 3A and details of exon sizes and intron-exon junctions are presented in Fig. 3B. Human PCTP consists of six exons and ¢ve introns. All intronexon junctions demonstrated GT/AG consensus splice sites. Despite the use of a long template PCR system (Boehringer Mannheim), we were unable to amplify a DNA fragment that spanned intron I. If we assume that the 10 and 3.5 kb EcoRI restriction fragments that contain exons 1 and 2, respectively, are adjacent to each other, the length of intron I could range from 3 to 13 kb (Fig. 3A). This suggests that PCTP spans 13^23 kb of the human genome. Exon sizes as well as intron-exon boundaries are very similar to the rat Pctp gene, which spans V10.8 kb of genomic DNA [8]. Results of 5PRACE (Fig. 1A) suggest that a transcription initiation site for PCTP is located 14 nucleotides (nt) upstream from the translation initiation site. However, when the nucleotide sequence from the 5PRACE cDNA fragment was utilized to search the EST database, a clone (GenBank accession number AA030013) was identi¢ed which contained an additional 95 bp of 5Puntranslated region (UTR). Whereas a single transcription initiation site was demonstrated for rat Pctp [8], this ¢nding suggests the possibility that a second transcription initiation site in the human gene is located 109 nt upstream to the translation initiation site or that the 5PRACE product does not represent a complete transcript. The chromosomal localization of PCTP was determined by radiation hybrid mapping [10] using the Genebridge4 Radiation Hybrid screening panel (Research Genetics). Primers (sense 5P-GACTCTGCACCTTTTTCTCAGG-3P, antisense 5P-CAAACAGTCTGAGGTATCCCAC-3P) were designed to the 3P untranslated region of human PC-TP cDNA (Fig. 1A) and were utilized to test samples of genomic DNA from each of the 93 radiation hybrid clones by PCR for the presence of a 210 bp PCTP-speci¢c
267
Fig. 2. Tissue-speci¢c expression of human PC-TP. Northern blot analysis of human poly-A RNA (2 Wg/lane) probed with 32 P-labeled human PC-TP cDNA (upper panels). Following autoradiography, the ¢lters were stripped and rehybridized with a 32 P-labeled human L-actin cDNA (lower panels).
DNA sequence. The results were tabulated and submitted to the Whitehead Institute/MIT Center for Genome Research (http://www-genome.wi.mit.edu/ cgi-bin/contig/rhmapper.pl). Based upon linkage to adjacent STS markers (Fig. 4A), the human PCTP gene was assigned to Chromosome 17, 6.08 cR from WI-5817 and 4.71 cR from WI-6277 (lod 2.66). Because WI-6277 maps to the interval D17S791 (65.0 cM)^D17S794 (84.2 cM), PCTP localizes to 17q2122 (www.ncbi.nlm.nih.gov/genemap98/). To facilitate mapping of Pctp in the mouse, we employed a rat liver PC-TP cDNA [8] to screen a mouse liver cDNA library (Stratagene). A 1689 bp cDNA clone encoding murine PC-TP was isolated and its nucleotide sequence was determined and deposited in GenBank with accession number AF114437. This clone contained a 399 bp incomplete ORF which was identical to the corresponding region of a partial cDNA (GenBank accession number Z50024) cloned from mouse lung [7]. However, the newly cloned cDNA also contained a complete 3PUTR which was utilized for genetic mapping by single strand conformation polymorphism (SSCP) analysis [11]. Oligonucleotide primers (sense 5P-CCTCGATGTTAGTAGGAAAAT-3P, antisense 5P-ACAGAACACAACTTTCTTCCC-3P) were designed based upon the 3P untranslated portion of the murine cDNA and employed for PCR ampli¢cation of
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Fig. 3. Structure of the human PCTP gene. (A) Schematic diagram in which introns and exons are represented by a solid lines and solid boxes, respectively. The EcoRI restriction endonuclease sites utilized for subcloning into pBluescript prior to nucleotide sequencing are indicated. Interruptions within the EcoRI fragment containing exon I indicate that intronic distances in this 10 kb segment of DNA are not drawn to scale. As described in the text, intron sizes were determined by PCR, with the exception of intron I for which `nd' indicates that no result was obtained. The bottom of the ¢gure presents a schematic representation of the human PC-TP cDNA indicating the contributions of each exon and the positions of the ORF and UTRs. (B) Characteristics of the six exons that comprise the human PCTP gene.
215 bp genomic DNA fragments from ¢ve inbred strains of mice (AKR/J, DBA/2J, C3H/HeJ, C57L/ J, C57BL/6J) and SPRET/Ei. Analysis of the PCR products by SSCP revealed a polymorphism in
SPRET/Ei compared with each of the inbred strains. Linkage between this polymorphism and previously mapped murine chromosomal markers was ascertained by SSCP analysis using the same PCR primers
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269
Fig. 4. Genetic mapping of PC-TP. (A) Radiation hybrid mapping of the human PCTP gene to chromosome 17q21-22. Genomic DNA samples from the 93 cell line Genebridge4 Radiation Hybrid screening panel were tested by PCR for the presence of a 210 bp PCTP-speci¢c nucleotide sequence. Results are presented for PCTP and for £anking STS markers from the database (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl), where 1 denotes presence of the indicated nucleotide sequence, 0 denotes its absence and 2 represents unknown/uncertain result. (B) Mapping of mouse Pctp to chromosome 11. (left side) Haplotype diagram of BSS progeny, where ¢lled boxes represent mice carrying a C57BL/6J allele. The number of o¡spring that carry each haplotype are given at the bottom of each column. (right side) Schematic representation of mouse chromosome 11 showing location of Pctp in relation to linked markers and genes. Recombination distances between loci are in cM.
and genomic DNA from a BSS panel consisting of 94 genotyped progeny of an interspeci¢c backcross [(C57BL/6JUSPRET/Ei)F1 USPRET/Ei] [12]. As illustrated in Fig. 4B, analysis of the resulting SSCP strain distribution pattern utilizing the Map Manager computer program [13] assigned murine Pctp to the region of syntenic conservation in the central portion of chromosome 11 (genetic distances in cM): centromere ^ D11Mit36 ^ Tbx ^ 3.3 þ 1.0 cM ^ Pctp ^ 1.1 þ 1.1 cM ^ Chad ^ 4.3 þ 2.1 cM ^ D11Mit10. Mapping of PCTP to human chromosome 17q2122 and mouse chromosome 11 should facilitate linkage of genetic disorders of lipid metabolism which could elucidate the function of PC-TP as well as its potential role in bile formation.
Acknowledgements This research was supported in part by a Pilot and Feasibility Grant (D.E. Cohen) from the Liver Research Center at Albert Einstein College of Medicine (DK41296, D.A. Shafritz, P.I.), NIH DK45639 (D.R. Beier) and the Program in Human Genetics at Albert Einstein College of Medicine. Support from the Alexandrine and Alexander L. Sinsheimer Fund (D.E. Cohen) is gratefully acknowledged. This work was performed while D.E. Cohen was a P¢zer Scholar in Cardiovascular Medicine and an American Liver Foundation Liver Scholar. The authors thank Ms. Holly Dushkin for expert technical assistance.
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References [1] K.W.A. Wirtz, Annu. Rev. Biochem. 60 (1991) 73^99. [2] D.W. Hay, M.C. Carey, Hepatology 12 (1990) 65^165. [3] T. Teerlink, T.P. van der Krift, M. Post, K.W.A. Wirtz, Biochim. Biophys. Acta 713 (1982) 61^67. [4] D.E. Cohen, M.R. Leonard, M.C. Carey, Biochemistry 33 (1994) 9975^9980. [5] W.W. LaMorte, M.L. Booker, S. Kay, Hepatology 28 (1998) 631^637. [6] D.E. Cohen, R.M. Green, Gene 163 (1995) 327^328. [7] T.B.H. Geijtenbeek, A.J. Smith, P. Borst, K.W.A. Wirtz, Biochem. J. 316 (1996) 49^55.
[8] M.K. Wu, M.O. Boylan, D.E. Cohen, Gene 235 (1999) 111^ 120. [9] M.S. Boguski, T.M. Lowe, C.M. Tolstoshev, Nat. Genet. 4 (1993) 332^333. [10] D.R. Cox, M. Burmeister, E.R. Price, S. Kim, R.M. Myers, Science 250 (1990) 245^250. [11] D.R. Beier, H. Dushkin, D.J. Sussman, Proc. Natl. Acad. Sci. USA 89 (1992) 9102^9106. [12] L.B. Rowe, J.H. Nadeau, R. Turner, W.N. Frankel, V.A. Letts, J.T. Eppig, M.S. Ko, S.J. Thurston, E.H. Birkenmeier, Mamm. Genome 5 (1994) 253^274. [13] K.F. Manley, R.W. Elliot, Mamm. Genome 1 (1991) 123^ 126.
BBAEXP 91309 19-10-99
Short sequence-paper
Cloning, tissue-speci¢c expression, gene structure and chromosomal localization of human phosphatidylcholine transfer protein1 David E. Cohen
a;
*, Richard M. Green b , Michele K. Wu a , David R. Beier
c
a
Departments of Medicine and Biochemistry, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Ullmann Building Room 625, 1300 Morris Park Avenue, Bronx, NY 10461, USA b Division of Digestive and Liver Diseases, University of Illinois at Chicago and West Side VAMC, 840 S. Wood St. M/C 787, Chicago, IL 60612, USA c Division of Genetics, Harvard Medical School, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA Received 29 June 1999; accepted 20 August 1999
Abstract Phosphatidylcholine transfer protein (PC-TP) is a cytosolic protein that catalyzes intermembrane transfer of phosphatidylcholines in vitro. We have cloned a cDNA encoding the human ortholog of PC-TP and have determined its tissue-specific expression as well as genomic organization. Radiation hybrid mapping localized the human gene, PCTP, to chromosome 17q21-22 and PCR-based single strand conformation polymorphism analysis of an interspecific backcross assigned mouse Pctp to the region of syntenic conservation on chromosome 11. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Phospholipid; Lipid; Transfer Protein; Liver; Bile
Phosphatidylcholine transfer protein (PC-TP) is a soluble protein that was ¢rst puri¢ed from bovine and rat liver more than two decades ago by virtue of its ability to catalyze intermembrane transfer of phosphatidylcholines exclusive of other phospholipid classes [1]. Although well characterized biochemically, the physiological function(s) of PC-TP remains unknown. Because phospholipids in bile are enriched ( s 95%) in phosphatidylcholines [2] and because PCTP is highly expressed in rat liver [3], we have hypothesized that PC-TP might function in hepatocel* Corresponding author. Fax: +1-718-430-8975; E-mail: [email protected] 1 The nucleotide sequence data in this paper have been submitted to GenBank and have been assigned accession numbers AF114430^AF114437.
lular selection and transport of phosphatidylcholines during bile formation [4]. In support of this possibility, we have shown that bile salts markedly stimulate in vitro activity of puri¢ed bovine liver PC-TP [4], and LaMorte et al. [5] have demonstrated recently that PC-TP displays highest a¤nities for the molecular species of phosphatidylcholines that are secreted into bile. Cloning of full-length or partial cDNAs has demonstrated that PC-TPs from cow [6,7], rat [7,8] and mouse [7] are highly homologous. To facilitate cDNA cloning of human PC-TP, the nucleotide sequence of a cDNA encoding bovine PC-TP [6] was utilized to search the expressed sequence tag (EST) database (http://www.ncbi.nlm.nih.gov/dbEST/) [9]. Clones with 5P and 3P nucleotide sequences similar to bovine PC-TP were identi¢ed (GenBank accession
0167-4781 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 9 ) 0 0 1 6 3 - 3
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Fig. 1. Nucleotide sequence of the 1932 bp cDNA encoding human PC-TP. Dots (...) indicate abbreviation of the 3P-untranslated region (UTR). Underlined nucleotide sequence was obtained from dbEST clones (GenBank accession numbers W02400 and N70264) as described in the text. The remainder of the nucleotide sequence was determined by 5PRACE and has been deposited in GenBank with accession number AF114430. The deduced 214 aa of the ORF are shown with the stop codon indicated by an asterisk (*).
numbers W02400 and N70264, respectively). The former clone was incomplete at its 5P end, and therefore rapid ampli¢cation of the 5P cDNA end (5PRACE) was employed in order to obtain a complete open reading frame (ORF). Because PC-TP mRNA is abundant in both mouse liver and kidney [7], human kidney cDNA (Clontech Marathon-Ready cDNA) was utilized as template for PCR together with gene speci¢c antisense primers (5P-CAAACAGTCTGAGGTATCCCAC-3P and 5P-TGAGGTAGTTCTGACAGGCTCTTGC-3P) designed to the dbEST nucleotide sequences. The nucleotide and deduced amino acid sequences of the composite cDNA encoding human PC-TP is presented in Fig. 1. Human PCTP is a 214 aa (24 842 Da) protein that is 76% and 80% identical to bovine [6,7] and rat [8] PC-TPs, respectively. In order to examine tissue-speci¢c expression, the cDNA from Fig. 1 was utilized to probe human multiple tissue Northern blots (Clontech). Fig. 2 demonstrates that highest PC-TP mRNA levels
were present in liver, placenta, testis, kidney and heart. Only thymus lacked evidence of PC-TP expression and low levels were observed in brain and lung. All other tissues demonstrated appreciable PC-TP mRNA levels. By comparison, Teerlink et al. [3] in an earlier study employed a radioimmunoassay to quantify PC-TP in rat tissues: Highest concentrations were found in liver followed by intestinal mucosa, kidney, spleen, lung and adrenals. In the adult mouse, expression of PC-TP mRNA [7] was highest in kidney, liver and testis, with low levels detected in most tissues and minimal levels in brain and thymus. In order to determine the structure of the human phosphatidylcholine transfer protein gene PCTP, the cDNA cloned by 5PRACE was utilized to screen ¢lters of a gridded RPCI Human PAC Library (RPCI1, Roswell Park Cancer Institute, Department of Human Genetics, Bu¡alo, NY, USA). Of the seven clones that hybridized (26L8, 30Mll, 30Tll, 77D6, 82M5, 108P13 and 296B16), Southern blot analysis of EcoRI digested DNA demonstrated that all but
BBAEXP 91309 19-10-99
D.E. Cohen et al. / Biochimica et Biophysica Acta 1447 (1999) 265^270
one (108P13) contained PCTP in its entirety. Hybridizing bands from one of the clones (296B16) were subcloned into pBluescript KS (Stratagene) for nucleotide sequence analysis. Utilizing primers designed to the cDNA, nucleotide sequences of exons as well as intron-exon junctions were determined. Intron sizes were determined by nucleotide sequencing and by PCR using exon-speci¢c primers. The structure of PCTP is presented schematically in Fig. 3A and details of exon sizes and intron-exon junctions are presented in Fig. 3B. Human PCTP consists of six exons and ¢ve introns. All intronexon junctions demonstrated GT/AG consensus splice sites. Despite the use of a long template PCR system (Boehringer Mannheim), we were unable to amplify a DNA fragment that spanned intron I. If we assume that the 10 and 3.5 kb EcoRI restriction fragments that contain exons 1 and 2, respectively, are adjacent to each other, the length of intron I could range from 3 to 13 kb (Fig. 3A). This suggests that PCTP spans 13^23 kb of the human genome. Exon sizes as well as intron-exon boundaries are very similar to the rat Pctp gene, which spans V10.8 kb of genomic DNA [8]. Results of 5PRACE (Fig. 1A) suggest that a transcription initiation site for PCTP is located 14 nucleotides (nt) upstream from the translation initiation site. However, when the nucleotide sequence from the 5PRACE cDNA fragment was utilized to search the EST database, a clone (GenBank accession number AA030013) was identi¢ed which contained an additional 95 bp of 5Puntranslated region (UTR). Whereas a single transcription initiation site was demonstrated for rat Pctp [8], this ¢nding suggests the possibility that a second transcription initiation site in the human gene is located 109 nt upstream to the translation initiation site or that the 5PRACE product does not represent a complete transcript. The chromosomal localization of PCTP was determined by radiation hybrid mapping [10] using the Genebridge4 Radiation Hybrid screening panel (Research Genetics). Primers (sense 5P-GACTCTGCACCTTTTTCTCAGG-3P, antisense 5P-CAAACAGTCTGAGGTATCCCAC-3P) were designed to the 3P untranslated region of human PC-TP cDNA (Fig. 1A) and were utilized to test samples of genomic DNA from each of the 93 radiation hybrid clones by PCR for the presence of a 210 bp PCTP-speci¢c
267
Fig. 2. Tissue-speci¢c expression of human PC-TP. Northern blot analysis of human poly-A RNA (2 Wg/lane) probed with 32 P-labeled human PC-TP cDNA (upper panels). Following autoradiography, the ¢lters were stripped and rehybridized with a 32 P-labeled human L-actin cDNA (lower panels).
DNA sequence. The results were tabulated and submitted to the Whitehead Institute/MIT Center for Genome Research (http://www-genome.wi.mit.edu/ cgi-bin/contig/rhmapper.pl). Based upon linkage to adjacent STS markers (Fig. 4A), the human PCTP gene was assigned to Chromosome 17, 6.08 cR from WI-5817 and 4.71 cR from WI-6277 (lod 2.66). Because WI-6277 maps to the interval D17S791 (65.0 cM)^D17S794 (84.2 cM), PCTP localizes to 17q2122 (www.ncbi.nlm.nih.gov/genemap98/). To facilitate mapping of Pctp in the mouse, we employed a rat liver PC-TP cDNA [8] to screen a mouse liver cDNA library (Stratagene). A 1689 bp cDNA clone encoding murine PC-TP was isolated and its nucleotide sequence was determined and deposited in GenBank with accession number AF114437. This clone contained a 399 bp incomplete ORF which was identical to the corresponding region of a partial cDNA (GenBank accession number Z50024) cloned from mouse lung [7]. However, the newly cloned cDNA also contained a complete 3PUTR which was utilized for genetic mapping by single strand conformation polymorphism (SSCP) analysis [11]. Oligonucleotide primers (sense 5P-CCTCGATGTTAGTAGGAAAAT-3P, antisense 5P-ACAGAACACAACTTTCTTCCC-3P) were designed based upon the 3P untranslated portion of the murine cDNA and employed for PCR ampli¢cation of
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Fig. 3. Structure of the human PCTP gene. (A) Schematic diagram in which introns and exons are represented by a solid lines and solid boxes, respectively. The EcoRI restriction endonuclease sites utilized for subcloning into pBluescript prior to nucleotide sequencing are indicated. Interruptions within the EcoRI fragment containing exon I indicate that intronic distances in this 10 kb segment of DNA are not drawn to scale. As described in the text, intron sizes were determined by PCR, with the exception of intron I for which `nd' indicates that no result was obtained. The bottom of the ¢gure presents a schematic representation of the human PC-TP cDNA indicating the contributions of each exon and the positions of the ORF and UTRs. (B) Characteristics of the six exons that comprise the human PCTP gene.
215 bp genomic DNA fragments from ¢ve inbred strains of mice (AKR/J, DBA/2J, C3H/HeJ, C57L/ J, C57BL/6J) and SPRET/Ei. Analysis of the PCR products by SSCP revealed a polymorphism in
SPRET/Ei compared with each of the inbred strains. Linkage between this polymorphism and previously mapped murine chromosomal markers was ascertained by SSCP analysis using the same PCR primers
BBAEXP 91309 19-10-99
D.E. Cohen et al. / Biochimica et Biophysica Acta 1447 (1999) 265^270
269
Fig. 4. Genetic mapping of PC-TP. (A) Radiation hybrid mapping of the human PCTP gene to chromosome 17q21-22. Genomic DNA samples from the 93 cell line Genebridge4 Radiation Hybrid screening panel were tested by PCR for the presence of a 210 bp PCTP-speci¢c nucleotide sequence. Results are presented for PCTP and for £anking STS markers from the database (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl), where 1 denotes presence of the indicated nucleotide sequence, 0 denotes its absence and 2 represents unknown/uncertain result. (B) Mapping of mouse Pctp to chromosome 11. (left side) Haplotype diagram of BSS progeny, where ¢lled boxes represent mice carrying a C57BL/6J allele. The number of o¡spring that carry each haplotype are given at the bottom of each column. (right side) Schematic representation of mouse chromosome 11 showing location of Pctp in relation to linked markers and genes. Recombination distances between loci are in cM.
and genomic DNA from a BSS panel consisting of 94 genotyped progeny of an interspeci¢c backcross [(C57BL/6JUSPRET/Ei)F1 USPRET/Ei] [12]. As illustrated in Fig. 4B, analysis of the resulting SSCP strain distribution pattern utilizing the Map Manager computer program [13] assigned murine Pctp to the region of syntenic conservation in the central portion of chromosome 11 (genetic distances in cM): centromere ^ D11Mit36 ^ Tbx ^ 3.3 þ 1.0 cM ^ Pctp ^ 1.1 þ 1.1 cM ^ Chad ^ 4.3 þ 2.1 cM ^ D11Mit10. Mapping of PCTP to human chromosome 17q2122 and mouse chromosome 11 should facilitate linkage of genetic disorders of lipid metabolism which could elucidate the function of PC-TP as well as its potential role in bile formation.
Acknowledgements This research was supported in part by a Pilot and Feasibility Grant (D.E. Cohen) from the Liver Research Center at Albert Einstein College of Medicine (DK41296, D.A. Shafritz, P.I.), NIH DK45639 (D.R. Beier) and the Program in Human Genetics at Albert Einstein College of Medicine. Support from the Alexandrine and Alexander L. Sinsheimer Fund (D.E. Cohen) is gratefully acknowledged. This work was performed while D.E. Cohen was a P¢zer Scholar in Cardiovascular Medicine and an American Liver Foundation Liver Scholar. The authors thank Ms. Holly Dushkin for expert technical assistance.
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D.E. Cohen et al. / Biochimica et Biophysica Acta 1447 (1999) 265^270
References [1] K.W.A. Wirtz, Annu. Rev. Biochem. 60 (1991) 73^99. [2] D.W. Hay, M.C. Carey, Hepatology 12 (1990) 65^165. [3] T. Teerlink, T.P. van der Krift, M. Post, K.W.A. Wirtz, Biochim. Biophys. Acta 713 (1982) 61^67. [4] D.E. Cohen, M.R. Leonard, M.C. Carey, Biochemistry 33 (1994) 9975^9980. [5] W.W. LaMorte, M.L. Booker, S. Kay, Hepatology 28 (1998) 631^637. [6] D.E. Cohen, R.M. Green, Gene 163 (1995) 327^328. [7] T.B.H. Geijtenbeek, A.J. Smith, P. Borst, K.W.A. Wirtz, Biochem. J. 316 (1996) 49^55.
[8] M.K. Wu, M.O. Boylan, D.E. Cohen, Gene 235 (1999) 111^ 120. [9] M.S. Boguski, T.M. Lowe, C.M. Tolstoshev, Nat. Genet. 4 (1993) 332^333. [10] D.R. Cox, M. Burmeister, E.R. Price, S. Kim, R.M. Myers, Science 250 (1990) 245^250. [11] D.R. Beier, H. Dushkin, D.J. Sussman, Proc. Natl. Acad. Sci. USA 89 (1992) 9102^9106. [12] L.B. Rowe, J.H. Nadeau, R. Turner, W.N. Frankel, V.A. Letts, J.T. Eppig, M.S. Ko, S.J. Thurston, E.H. Birkenmeier, Mamm. Genome 5 (1994) 253^274. [13] K.F. Manley, R.W. Elliot, Mamm. Genome 1 (1991) 123^ 126.
BBAEXP 91309 19-10-99