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Cloning and characterization of a cDNA encoding alkaline phosphatase in mouse embryonal carcinoma cells
Clinica Chimica Actu, 186 (1989) 171-174 Elsevier
171
CCA 04587
Short Communication
Cloning and characterization of a cDNA encoding alkaline phosphatase in mouse embryonal carcinoma cells Ann C. Hahnel and Gilbert A. Schultz Department
of Medical Biochemistry,
University of Calgary
Calgary, Alberta (Canada)
(Received 14 June 1989; accepted 7 August 1989) Key words: Alkaline phosphatase; cDNA library; DNA sequence analysis; Embryonal carcinoma cells
Introduction
Alkaline phosphatases (APs) are membrane-bound, glycosylated, homodimeric enzymes that are present on the surfaces of many different cell types in the body. In man they are encoded by four different genes that represent the isozyme forms characteristic of germ cell, placenta, intestine and tissue non-specific (or liver/ bone/kidney) AP, respectively [l]. While high levels of AP activity are present in adult mouse bone, liver, intestine, kidney and placenta [2], activity is also prominent in preimplantation embryos, embryonal carcinoma (EC) cells and migrating primordial germ cells [3-61. A first step toward the eventual understanding of the regulation of expression of AP genes and the possible role of AP gene expression during early embryogenesis requires the cloning and characterization of the specific genes within the small multi-gene family that are expressed. To this end we have screened a cDNA library constructed from poly (A) + RNA of EC cells for clones containing AP sequences. A full-length cDNA coding for a liver/bone/kidney-type of alkaline phosphatase has been isolated and sequenced. Materials and methods
RNA was extracted from Nulli SCCl EC cells [7] and poly A( + ) RNA was purified by two passages over an oligo-dT cellulose affinity column. The poly A( + ) RNA fraction was used to generate double stranded cDNA molecules and a cDNA library was constructed in hgtll by procedures described previously [8]. Putative AP cDNAs were selected from the Xgtll clones by hybridization with two synthetic oligonucleotides derived from the rat osteosarcoma AP cDNA sequence [9, nucleoCorrespondence and requests for reprints to: Dr. Gilbert A. Schultz, Department of Medical Biochemistry, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4Nl Canada.
0009-8981/89/$03.50
0 1989 Elsezier Science Publishers B.V. (Biomedical Division)
172
tides 1143-1181 and nucleotides 1269-13041 that are within highly conserved regions of all AP cDNA clones sequenced to date [l]. Clones containing 2.4 kb and 1.4 kb inserts were transferred to the vector Bluescript (Stratagene), deletion clones were constructed by exonuclease III digestion and sequencing was carried out by a modified dideoxy method [lo]. Results
and discussion
There were lo6 independent clones in the Xgtll cDNA library constructed from EC cell mRNA. The library was amplified and approximately 2 X lo5 plaque-forming units were screened with the two oligonucleotides derived from the rat liver/ bone/kidney AP sequence. The two plaques that were clearly positive with both probes were cloned, and the inserts were found to be 2.4 and 1.4 kb long. The sequence of the 2.4 kb AP cDNA clone is presented in Fig. 1. The sequence of the 1.4 kb clone confirmed the longer sequence beginning with nucleotide 919 and added an additional eight nucleotides to the 3’-end. The putative coding sequence
Fig. 1. Nucleotide sequence of a 2.4 kb cDNA clone derived from Nulli SCCl cells. Brackets at the 3’-end indicate additional nucieotides from a 1.4 kb clone. Differences in the 3’-untranslated region of Nulli SCCl and placental APs are shown by a single underline, differences in amino acids are in boxes; putative signal sequence is indicated by dashed line, active site serine by asterisk, polyadenylation signal by heavy underline and asparagine-linked glycosylation sites by arrowheads.
173
predicts a core protein of 524 amino-acids with a mol wt of 57 kDa. The sequence of the AP cDNA from Nulli SCCl cells differs from another mouse AP clone isolated from a placental cDNA library [ll] by three minor changes in the 3’untranslated region and by two nucleotides in the coding region that alter an amino acid at each end of the protein. The placental and EC cell cDNAs were derived from different mouse strains. We do not know if these differences are indicative of two closely related genes or of allelic variants between strains. In either case, both messages code for a protein that is 90% identical, at the amino-acid level, to the human liver/bone/kidney enzyme [12] and only 55% identical to the human intestinal form [13] and 57% identical to the human germ cell isozyme [14]. Human placental AP is anchored to the cytoplasmic membrane by a phosphatidylinositol glycan moiety that is attached to aspartic acid residue 484 by post-translational mechanisms [15]. The mouse AP from EC cells, like other mouse APs [l], has a serine residue at the equivalent position in the polypeptide chain. Thus, if the cell membrane anchoring mechanism for mouse AP also utilizes a phosphatidylinositol glycan linkage, it must involve either a different amino acid or a different site of attachment than that in human placental AP. Acknowledgements
This work was supported by grant MT4851 from the Medical Research Council of Canada. A.C.H. was the recipient of a postdoctoral fellowship from the Alberta Heritage Foundation for Medical Research. References 1 Millan JL. Gncodevelopmental expression and structure of alkaline phosphatase genes. Anticancer Res 1988;8:995-1004. 2 Goldstein DJ, Rogers CE, Harris H. Expression of alkaline phosphatase loci in mammahan tissues. Proc Nat1 Acad Sci USA 1980;77:2857-2860. 3 Johnson LV, Calarco PG, Siebert ML. Alkaline phosphatase activity in preimplantation mouse embryos. J Embryo1 Exp Morph 1977;14:83-89. 4 Bernstine EG, Hooper ML, Grandchamp S, Ephrussi B. Alkaline phosphatase activity in mouse teratoma. Proc Nat1 Acad Sci USA 1973;70:3899-3903. 5 Chiquone AD. The identification, origin and migration of the primordial germ cells in the mouse embryo. Anat Ret 1954;118:135-146. 6 Mintr B, Russell ES. Gene-induced embryological modifications of primordial germ cells in the mouse. J Exp Zoo1 1957;134:207-238. 7 Hahnel AC, Gifford DJ, Heildcila JJ, Schultz GA. Expression of the major heat shock protein (hsp 70) family during early mouse development. Terat Cam Mut 1986;6:493-510. 8 Hagan FS, Gray CL, Kuijper JL. Assaying the quality of cDNA libraries. BioTechniques 1988;6:340-345. 9 Thiede MA, Yoon K, Golub EE, Noda M, Rodan GA. Structure and expression of rat osteosarcoma (ROS 17/2.8) alkaline phosphatase: product of a single copy gene. Proc Nat1 Acad Sci USA 1988;85:319-323. 10 Johnston-Dow L, Mardis E, Heiner C, Roe BA. Optimized methods for fluorescent and radiolabeled DNA sequencing. BioTechniques 1987;5:754-765.
174 11 Terao M, Mintz B. Cloning and characterization
12
13 14 15
of a cDNA coding for mouse placental alkaline phosphatase. Proc Nat1 Acad Sci USA 1987;84:7051-7055. Weiss MJ, Henthom PS, Lafferty MA, Slaughter C, Raducha M, Harris H. Isolation and characterization of a cDNA encoding a human liver/bone/kidney-type alkaline phosphatase. Proc Nat1 Acad Sci USA 1986;83:7182-7186. Berger J, Garattini E, Hua J-C, Udenfriend S. Cloning and sequencing of human intestinal alkaline phosphatase cDNA. Proc Nat1 Acad Sci USA 1987;84:695-698. Millan JL, Manes T. Seminoma-derived Nagao isozyme is encoded by a germ-cell alkaline phosphatase gene. Proc Nat1 Acad Sci USA 1988;85:3024-3028. Micanovic R, Bailey CA, Brink L, Gerber L, Pan YCE, Hulmes JD, Udenfriend S. Aspartic acid-484 of nascent placental alkaline phosphatase condenses with a phosphatidyl inositol glycan to become the carboxyl terminus of the mature enzyme. Proc Natl Acad Sci USA 1988;85:1398-1402.
171
CCA 04587
Short Communication
Cloning and characterization of a cDNA encoding alkaline phosphatase in mouse embryonal carcinoma cells Ann C. Hahnel and Gilbert A. Schultz Department
of Medical Biochemistry,
University of Calgary
Calgary, Alberta (Canada)
(Received 14 June 1989; accepted 7 August 1989) Key words: Alkaline phosphatase; cDNA library; DNA sequence analysis; Embryonal carcinoma cells
Introduction
Alkaline phosphatases (APs) are membrane-bound, glycosylated, homodimeric enzymes that are present on the surfaces of many different cell types in the body. In man they are encoded by four different genes that represent the isozyme forms characteristic of germ cell, placenta, intestine and tissue non-specific (or liver/ bone/kidney) AP, respectively [l]. While high levels of AP activity are present in adult mouse bone, liver, intestine, kidney and placenta [2], activity is also prominent in preimplantation embryos, embryonal carcinoma (EC) cells and migrating primordial germ cells [3-61. A first step toward the eventual understanding of the regulation of expression of AP genes and the possible role of AP gene expression during early embryogenesis requires the cloning and characterization of the specific genes within the small multi-gene family that are expressed. To this end we have screened a cDNA library constructed from poly (A) + RNA of EC cells for clones containing AP sequences. A full-length cDNA coding for a liver/bone/kidney-type of alkaline phosphatase has been isolated and sequenced. Materials and methods
RNA was extracted from Nulli SCCl EC cells [7] and poly A( + ) RNA was purified by two passages over an oligo-dT cellulose affinity column. The poly A( + ) RNA fraction was used to generate double stranded cDNA molecules and a cDNA library was constructed in hgtll by procedures described previously [8]. Putative AP cDNAs were selected from the Xgtll clones by hybridization with two synthetic oligonucleotides derived from the rat osteosarcoma AP cDNA sequence [9, nucleoCorrespondence and requests for reprints to: Dr. Gilbert A. Schultz, Department of Medical Biochemistry, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4Nl Canada.
0009-8981/89/$03.50
0 1989 Elsezier Science Publishers B.V. (Biomedical Division)
172
tides 1143-1181 and nucleotides 1269-13041 that are within highly conserved regions of all AP cDNA clones sequenced to date [l]. Clones containing 2.4 kb and 1.4 kb inserts were transferred to the vector Bluescript (Stratagene), deletion clones were constructed by exonuclease III digestion and sequencing was carried out by a modified dideoxy method [lo]. Results
and discussion
There were lo6 independent clones in the Xgtll cDNA library constructed from EC cell mRNA. The library was amplified and approximately 2 X lo5 plaque-forming units were screened with the two oligonucleotides derived from the rat liver/ bone/kidney AP sequence. The two plaques that were clearly positive with both probes were cloned, and the inserts were found to be 2.4 and 1.4 kb long. The sequence of the 2.4 kb AP cDNA clone is presented in Fig. 1. The sequence of the 1.4 kb clone confirmed the longer sequence beginning with nucleotide 919 and added an additional eight nucleotides to the 3’-end. The putative coding sequence
Fig. 1. Nucleotide sequence of a 2.4 kb cDNA clone derived from Nulli SCCl cells. Brackets at the 3’-end indicate additional nucieotides from a 1.4 kb clone. Differences in the 3’-untranslated region of Nulli SCCl and placental APs are shown by a single underline, differences in amino acids are in boxes; putative signal sequence is indicated by dashed line, active site serine by asterisk, polyadenylation signal by heavy underline and asparagine-linked glycosylation sites by arrowheads.
173
predicts a core protein of 524 amino-acids with a mol wt of 57 kDa. The sequence of the AP cDNA from Nulli SCCl cells differs from another mouse AP clone isolated from a placental cDNA library [ll] by three minor changes in the 3’untranslated region and by two nucleotides in the coding region that alter an amino acid at each end of the protein. The placental and EC cell cDNAs were derived from different mouse strains. We do not know if these differences are indicative of two closely related genes or of allelic variants between strains. In either case, both messages code for a protein that is 90% identical, at the amino-acid level, to the human liver/bone/kidney enzyme [12] and only 55% identical to the human intestinal form [13] and 57% identical to the human germ cell isozyme [14]. Human placental AP is anchored to the cytoplasmic membrane by a phosphatidylinositol glycan moiety that is attached to aspartic acid residue 484 by post-translational mechanisms [15]. The mouse AP from EC cells, like other mouse APs [l], has a serine residue at the equivalent position in the polypeptide chain. Thus, if the cell membrane anchoring mechanism for mouse AP also utilizes a phosphatidylinositol glycan linkage, it must involve either a different amino acid or a different site of attachment than that in human placental AP. Acknowledgements
This work was supported by grant MT4851 from the Medical Research Council of Canada. A.C.H. was the recipient of a postdoctoral fellowship from the Alberta Heritage Foundation for Medical Research. References 1 Millan JL. Gncodevelopmental expression and structure of alkaline phosphatase genes. Anticancer Res 1988;8:995-1004. 2 Goldstein DJ, Rogers CE, Harris H. Expression of alkaline phosphatase loci in mammahan tissues. Proc Nat1 Acad Sci USA 1980;77:2857-2860. 3 Johnson LV, Calarco PG, Siebert ML. Alkaline phosphatase activity in preimplantation mouse embryos. J Embryo1 Exp Morph 1977;14:83-89. 4 Bernstine EG, Hooper ML, Grandchamp S, Ephrussi B. Alkaline phosphatase activity in mouse teratoma. Proc Nat1 Acad Sci USA 1973;70:3899-3903. 5 Chiquone AD. The identification, origin and migration of the primordial germ cells in the mouse embryo. Anat Ret 1954;118:135-146. 6 Mintr B, Russell ES. Gene-induced embryological modifications of primordial germ cells in the mouse. J Exp Zoo1 1957;134:207-238. 7 Hahnel AC, Gifford DJ, Heildcila JJ, Schultz GA. Expression of the major heat shock protein (hsp 70) family during early mouse development. Terat Cam Mut 1986;6:493-510. 8 Hagan FS, Gray CL, Kuijper JL. Assaying the quality of cDNA libraries. BioTechniques 1988;6:340-345. 9 Thiede MA, Yoon K, Golub EE, Noda M, Rodan GA. Structure and expression of rat osteosarcoma (ROS 17/2.8) alkaline phosphatase: product of a single copy gene. Proc Nat1 Acad Sci USA 1988;85:319-323. 10 Johnston-Dow L, Mardis E, Heiner C, Roe BA. Optimized methods for fluorescent and radiolabeled DNA sequencing. BioTechniques 1987;5:754-765.
174 11 Terao M, Mintz B. Cloning and characterization
12
13 14 15
of a cDNA coding for mouse placental alkaline phosphatase. Proc Nat1 Acad Sci USA 1987;84:7051-7055. Weiss MJ, Henthom PS, Lafferty MA, Slaughter C, Raducha M, Harris H. Isolation and characterization of a cDNA encoding a human liver/bone/kidney-type alkaline phosphatase. Proc Nat1 Acad Sci USA 1986;83:7182-7186. Berger J, Garattini E, Hua J-C, Udenfriend S. Cloning and sequencing of human intestinal alkaline phosphatase cDNA. Proc Nat1 Acad Sci USA 1987;84:695-698. Millan JL, Manes T. Seminoma-derived Nagao isozyme is encoded by a germ-cell alkaline phosphatase gene. Proc Nat1 Acad Sci USA 1988;85:3024-3028. Micanovic R, Bailey CA, Brink L, Gerber L, Pan YCE, Hulmes JD, Udenfriend S. Aspartic acid-484 of nascent placental alkaline phosphatase condenses with a phosphatidyl inositol glycan to become the carboxyl terminus of the mature enzyme. Proc Natl Acad Sci USA 1988;85:1398-1402.