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Sequence and Expression of the Mouse Homologue to Human Phospholipase C β3 Neighboring Gene
JOBNAME: BBRC 223#2 PAGE: 1 SESS: 8 OUTPUT: Wed Jun 19 13:39:57 1996 /xypage/worksmart/tsp000/72170k/24
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
223, 335–340 (1996)
0895
Sequence and Expression of the Mouse Homologue to Human Phospholipase C b3 Neighboring Gene1 Jacob Lagercrantz,*,2,3 Darek Kedra,*,3 Emma Carson,* Magnus Nordenskjöld,* Jan P. Dumanski,* Günther Weber,* and Fredrik Piehl† *Department of Molecular Medicine, Clinical Genetics Unit, Karolinska Hospital, S-171 76 Stockholm, Sweden; and †Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden Received May 3, 1996 We describe the isolation and expression of a murine homologue of the Phospholipase C b3 Neighboring Gene (PNG), located in the MEN1 region on chromosome 11q13. The PNG cDNA was isolated using a human PNG cDNA clone (SOM172). Human and mouse PNG do not have any marked similarity to other known genes on the DNA level, but the predicted protein display similarity to the C-terminal part of Phospholipase C b2. Northern blots with mouse PNG probes revealed expression of a 1 kb message in multiple tissues, and an additional 2.3 kb band in testis. The predicted murine protein contains 203 amino acids. In situ hybridization histochemistry displayed png mRNA expression in several tissues of the midstage mouse embryo, including the central nervous system. In late stage embryos, png was highly expressed in skeletal muscle, retina and neocortex. In the adult animal, expression was restricted to testis and thymus. © 1996 Academic Press, Inc.
We have earlier described the isolation, characterization and genomic structure of a human gene, Phospholipase C b3 Neighboring Gene (PNG), located in the Multiple Endocrine Neoplasia type 1 (MEN1) region on chromosome 11q13. The cDNA was isolated using the cosmid cCLGW4/ D11S750 which also contains the phospholipase C b3 gene (PLCB3) (1) and the VEGF Related Factor (VRF) (2,3). Human PNG does not have any marked similarity to other known genes on the DNA level. PNG showed expression of a 1 kb message in multiple tissues. The predicted protein of 199 amino acids is dominated by glycine, proline and alanine in the N-terminal half and shows similarity to collagen-like proteins. The gene spans approximately 2.5 kb and is divided into 4 exons and 3 introns, and it is located 4.4 kb upstream of the PLCb3 gene, with the 59 ends facing each other. The intergenic region has been completely sequenced, revealing separate CpG islands at both ends of this region. The islands are separated by a stretch of 2 kb, characterized by periodical alteration of the GC content. The 59 flanking region of PNG does not contain TATA or CCAAT boxes which would be in agreement with a house keeping promoter structure (4). To further characterize PNG and to evaluate its status as a MEN1 candidate gene we have cloned and sequenced the murine homologue (png) and investigated its expression pattern in pre- and postnatal mice with in situ hybridization histochemistry. MATERIAL AND METHODS Isolation of cDNAs. Murine png clones were selected from a lambda Zap new born whole brain cDNA library (Stratagene). Primary phages from high density filters (5 × 104 pfu/plate) were identified by hybridization with the 32P-labeled human PNG cDNA (pSOM172). Hybridization and washes of nitrocellulose membranes (Schleicher & Schüll) were carried out at 65°C under stringent conditions. Positive plaques were picked, purified and excised in vivo to produce bacterial colonies containing cDNA clones in pBluescript SK-. Nucleotide sequencing and analysis. Plasmid DNA was mini-prepared through alkaline lysis (5). Plasmid insert DNA was 1
The sequences reported in this paper have been deposited in the EMBL data base (Accession No. X97490). To whom correspondence should be addressed. Fax: +46 8 327734. 3 J.L. and D.K. contributed equally to the work presented here. Abbreviations: PNG, Phospholipase C b3 Neighboring Gene; MEN1, Multiple Endocrine Neoplasia type 1; nt, nucleotide; CNS, central nervous system. 2
335 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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mechanically sheared using a nebulizer (IPI Medical Products, USA no. 4207) (6). DNA fragments between 500–600 bp were blunt-end cloned into pUC18 vector (Pharmacia). Taq Dye Deoxy Terminators kit (ABI) was used for cycle sequencing. The sequences were assembled using the Staden program package (7). Database searches and sequence comparisons were done using FastA and gap programs from Wisconsin Package, Genetics Computer Group, WI, USA. Animals. Timed pregnant (n44) and young adult (n42) mice (C57 inbred strain, ALAB, Sweden) were sacrificed with carbon dioxide, and the relevant tissues were taken out and frozen on a chuck. Tissues were kept at −70°C until further use. Three gestational ages were used in this study; embryonic day 8, (E8), E14 and E17. All animal experiments in this study were approved by the local ethics committee for animal experimentation. In situ hybridization histochemistry. In situ hybridization was performed as previously described (8). Briefly, transverse sections (14 mm) were cut in a cryostat (Microm, Germany), thawed onto Probe-On slides (Fisher Scientific, USA) and stored in black, sealed boxes at −70°C until used. The sequences of the synthetic oligonucleotides complementary to mRNA encoding png were CAGGGGGACT CTGGAAGTAG ACTCGGGGCC CTGTGCTGCC A (complementary to nt 327– 367) and CCCGTCAGTT TAATAACAAT AAAAAACCCC AAAAGTGGAA AACTGAGGG (complementary to nt 855–903). The probes were labeled at the 39-end with deoxyadenosine-alpha[thio]triphosphate[35S] (NEN, USA) using terminal deoxynucleotidyl transferase (IBI, USA) to a specific activity of 7–10 × 108 cpm/mg and hybridized to the sections without pretreatment for 16–18 h at 42°C. The hybridization mixture contained: 50% formamide, 4 × SSC (1 × SSC40.15 M NaCl and 0.015 M sodium-citrate), 1 × Denhardt’s solution (0.02% each of polyvinyl-pyrrolidone, BSA and Ficoll), 1% sarcosyl (N-lauroylsarcosine; Sigma, USA), 0.02 M phosphate buffer (pH 7.0), 10% dextran sulfate (Pharmacia, Sweden), 250 mg/ml yeast tRNA (Sigma), 500 mg/ml sheared and heat denatured salmon sperm DNA (Sigma) and 200 mM dithiothreitol (DTT; LKB, Sweden). In control sections, the specificity of both probes was checked by adding a 20-fold excess of unlabeled probe to the hybridization mixture. In addition adjacent sections were hybridized with probes unrelated to PNG that gave different expression patterns. Following hybridization the sections were washed several times in 1×SSC at 55°C, dehydrated in ethanol and dipped in NTB2 nuclear track emulsion (Kodak, USA) and cover slipped. In some cases, sections were exposed to autoradiographic film (Beta-max autoradiography film Amersham Ltd, UK) prior to emulsion dipping.
RESULTS Mouse png homologues were isolated by screening a murine cDNA library with a human PNG cDNA clone. Five clones of sizes varying from 0.8–1 kb were recovered and sequenced. The cDNA sequences were compiled to give a full length 980 bp cDNA sequence covering the entire open reading frame (609 bp and 39 UTR (275 bp), as well as 96 bp of the 59 UTR. The overall homology of mouse png to human PNG predicted open reading frames were 86% identity and 91% similarity, respectively (fig. 1). The nucleotide sequence were more than 90% identical over the entire coding region. The longest predictable open reading frame of png encodes 203 amino acid residues (fig. 1). The average GC content of the first 300 bp of mouse and human PNG ranges between 80 and 85%, even
FIG. 1. Comparison of predicted amino acid sequences of the human and mouse PNG gene. Amino acid positions are on the left side. 336
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extending into the coding region. From the first 100 predicted amino acids, 65 are alanine, glycine, or proline (25, 25, and 15, respectively). This is also a feature of different types of collagen like genes. Consequently a database search recognized homologies with genes which share this characteristic (not shown). The database search did also identify a close to perfect match with two expressed sequence tags. The first one, H39427, is at rat cDNA clone identified when screening for upregulated genes in astrocytes treated with interferon gamma. The second one, D18307, is a mouse cDNA clone detected when sequencing genes expressed in mouse embryogenesis. In addition a FastA search of the Swissprot protein sequence database, using the predicted protein sequence of png, detected a 28% identity and 74% similarity with the C-terminal region (position 960–1060) of Phospholipase C b2. Protein prediction analysis (9) showed a high probability for a turned structure between amino acid residues 33–44, 74–81 and 89–105. The N- and C-terminal regions are predicted to contain mainly helical structures. Northern blot analysis of RNA from adult mouse tissues (heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis) showed expression in all tissues. The strongest expression was seen in testis, which also had an additional band (fig. 2). This is somewhat different to the pattern observed for human PNG in which similar levels of expression have been identified in all tissues examined (4). The 1 kb murine message presumably corresponds to the shorter of the two testis’ transcripts. The two png oligonucleotide probes gave identical hybridization patterns in all examined tissues. Mouse png expression was detected in the 8 day old embryos (E8), in which positive signals were recorded over structures most likely corresponding to the neuronal tube (not shown). In sagittal sections of the E14 mouse embryo, a positive hybridization signal was present over large parts of the brain, spinal cord, heart, liver, intestines and tongue (fig. 3A). At a later gestational age, E17, the hybridization signal was markedly reduced, except in skeletal muscle, thymus and neocortex
FIG. 2. Autoradiogram of a multiple tissue Northern blot hybridized with mouse png cDNA and GAPDH as a positive control. In all tissues tested a single transcript of 1 kb is detected. In testis an additional band of 2.3 kb is also found. Sizes of RNA markers are given in kb. 337
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FIG. 3. In situ hybridization autoradiographs (A, B) and dark field micrographs (C–F) showing the expression of PNG mRNA in the mouse. Multiple tissues, including the brain (Cx), spinal cord (Sc), heart (Ha), liver (L), intestines (I) and tongue (T), display a positive hybridization signal in the E14 embryo (A). In the E17 embryo (B), PNG is expressed preferably in skeletal muscle (M), neocortex (Cx) and thymus (Th). A strong signal is also recorded over the retina and, to a lesser degree, other parts of the eye in the E17 embryo (C). In the early postnatal mouse (D), a positive signal is present over the thymus (Th), while the heart (Ha) show no or weak labeling. PNG is expressed in the adult mouse testis (E), and grain clusters are shown, in toloudine counterstained sections, over cell body profiles in the tubuli seminiferi (F). Scale bars 4 0.8mm (A), 1.6mm (B), 0.2mm (C), 0.3mm (D), 0.25mm (E), 0.1mm (F). 338
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(fig. 3B). A strong hybridization signal was also recorded over retina (fig. 3C). Most tissues, including the pancreas and the suprarenal, pituitary and thyroid glands, displayed low or no specific labeling in the adult mouse. However, a positive signal was present over the thymus in the early postnatal mouse (fig. 3D) and over adult mouse testis (fig. 3E). In sections counterstained with toluidine, grain clusters were present over cell profiles constituting the tubuli seminiferi (fig. 3F). No specific labeling over background signal was recorded in control sections hybridized with an excess of cold probe (not shown). DISCUSSION Our attempts to characterize genes in the region for Multiple Endocrine Neoplasia type 1 on chromosome 11q13 (10) resulted in the isolation of a number of different transcripts (11). One of them was a 1 kb cDNA clone identified with the cosmid cCLGW4/D11S750, which we previously had isolated from the region (12). The gene was named Phospholipase Neighboring Gene (PNG) due to its close proximity to the Phospholipase C b3 gene (4). Using the human PNG cDNA clone pSOM172 we subsequently isolated and sequenced the mouse homologue of PNG (png). The transcript, 980 bp, of the longest clone has approximately the same size as the transcript determined from Northern blot analysis (fig. 2). The high similarity between the mouse and human PNG (91%) predicted open reading frames suggests that this cDNA clone indeed represents the murine counterpart of the human PNG gene. A region in the N-terminal part of the predicted protein does not display a significant homology with its human analog. Still, on the nucleotide level this region maintains a high homology with human PNG. This discrepancy could be due to an error in one of the two sequences. If so, the quality of the sequences suggests that it is most likely the human one. It is interesting to note that a Swissprot FastA search of the png predicted protein detects a similarity with the Phospholipase C b2 gene, in the region interacting with the Gqa G-protein subunit (13). If this similarity reflects a functional property of PNG it could indicate a regulatory relationship between PNG and the neighboring gene PLCB3 (4). Since MEN1 is generally believed to be a tumor suppressor gene, it seems reasonable to assume that it has normally to be expressed in tissues affected by MEN1 disease, to be considered a candidate gene for this disease. However, it may be that this expression is only seen during specific stages of development. To test the relevance of PNG as a candidate gene for MEN1 we have here investigated its expression during mouse development in general and in endocrine organs in particular. In situ hybridization histochemistry revealed a high png expression in multiple tissues of the midstage mouse embryo, with the highest expression in the developing CNS. Later in prenatal development, png expression was confined preferably to skeletal muscle and the neocortex, while the expression in many of the internal organs and parts of the CNS had been down-regulated. Thus, the expression profile of PNG may be consistent with a functional role during phases of organogenesis characterized by rapid growth. Interestingly, in the postnatal mouse, PNG expression was confined mainly to the thymus and testis, two types of tissue where cell proliferation is very high also in the adult animal. It is also noteworthy that, in the context of testis and thymus expression, thymic carcinoids and infertility have been shown to be involved in cases of MEN1 disease (14, 15). However, we were not able to detect any significant levels of png expression in other organs more commonly affected by MEN1, such as the pituitary. All together, this argues that PNG is not an ideal candidate gene for MEN1. ACKNOWLEDGMENTS This work was supported by grants from the Swedish Cancer Society, the Swedish Medical Research Council, the Magnus Bergvall Foundation, Markus Borgströms Fund, and The Cancer Society in Stockholm. 339
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REFERENCES 1. Lagercrantz, J., Carson, E., Phelan, C., Grimmond, S., Rosén, A., Daré, E., Nordenskjöld, M., Hayward, N. K., Larsson, C., and Weber, G. (1995) Genomics 26, 467–472. 2. Grimmond, S., Lagercrantz, J., Drinkwater, C., Silins, G., Townson, S., Pollock, P., Gotley, D., Carson, E., Nordenskjöld, M., Ward, L., Hayward, N., and Weber, G. (1996) Genome Research 6, 124–131. 3. Townson, S., Lagercrantz, J., Silins, G., Grimmond, S., Weber, G., and Hayward, N. (1996) Biochem. Biophys. Res. Com. 220, 922–928. 4. Lagercrantz, J., Carson, E., Larsson, C., Nordenskjöld, M., and Weber, G. (1996) Genomics 31, 380–384. 5. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 6. Pan, H.-Q., Wang, Y.-P., Chissoe, S. L., Bodenteich, A., Wang, Z., Iyer, K., Clifton, S. W., Crabtree, J. S., and Roe, B. A. (1994) GATA 11, 181–186. 7. Griffin, A. M., and Griffin, H. G. (1994) The Staden Package, Methods in Molecular Biology, pp. 9–170, Humana Press, Totawa, NJ. 8. Dagerlind, A., Friberg, K., Bean, A. J., and Hökfelt, T. (1992) Histochemistry 98, 39–49. 9. Chou, P. Y., and Fasman, G. D. (1978) Ann. Rev. Biochem. 47, 251–276. 10. Larsson, C., Skogseid, B., Öberg, K., Nakamura, Y., and Nordenskjöld, M. (1988) Nature 332, 85–87. 11. Lagercrantz, J., Larsson, C., Grimmond, S., Skogseid, B., Gobl, A., Friedman, E., Carson, E., Phelan, C., Öberg, K., Nordenskjöld, N., Hayward, N. K., and Weber, G. (1995) J. Int. Med. 238, 245–248. 12. Larsson, C., Weber, G., Kvanta, E., Lewis, K., Janson, M., Jones, C., Glaser, T., Evans, G., and Nordenskjöld, M. (1992) Hum. Genet. 89, 187–193. 13. Wu, D., Jiang, H., Katz, A., and Simon, M. I. (1993) J. Biol. Chem. 268, 3704–3709. 14. Teh, B. T., Hayward, N. K., Walters, M. K., Shepherd, J. J., Wilkinson, S., Nordenskjöld, M., and Larsson, C. (1994) J. Med. Genet. 31, 261–262. 15. Brandi, M. L., Weber, G., Svensson, A., Falchetti, A., Tonelli, F., Castello, R., Furlani, L., Scappaticci, S., Fraccaro, M., and Larsson, C. (1993) American Journal of Hum. Genet. 53, 1167–72.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
223, 335–340 (1996)
0895
Sequence and Expression of the Mouse Homologue to Human Phospholipase C b3 Neighboring Gene1 Jacob Lagercrantz,*,2,3 Darek Kedra,*,3 Emma Carson,* Magnus Nordenskjöld,* Jan P. Dumanski,* Günther Weber,* and Fredrik Piehl† *Department of Molecular Medicine, Clinical Genetics Unit, Karolinska Hospital, S-171 76 Stockholm, Sweden; and †Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden Received May 3, 1996 We describe the isolation and expression of a murine homologue of the Phospholipase C b3 Neighboring Gene (PNG), located in the MEN1 region on chromosome 11q13. The PNG cDNA was isolated using a human PNG cDNA clone (SOM172). Human and mouse PNG do not have any marked similarity to other known genes on the DNA level, but the predicted protein display similarity to the C-terminal part of Phospholipase C b2. Northern blots with mouse PNG probes revealed expression of a 1 kb message in multiple tissues, and an additional 2.3 kb band in testis. The predicted murine protein contains 203 amino acids. In situ hybridization histochemistry displayed png mRNA expression in several tissues of the midstage mouse embryo, including the central nervous system. In late stage embryos, png was highly expressed in skeletal muscle, retina and neocortex. In the adult animal, expression was restricted to testis and thymus. © 1996 Academic Press, Inc.
We have earlier described the isolation, characterization and genomic structure of a human gene, Phospholipase C b3 Neighboring Gene (PNG), located in the Multiple Endocrine Neoplasia type 1 (MEN1) region on chromosome 11q13. The cDNA was isolated using the cosmid cCLGW4/ D11S750 which also contains the phospholipase C b3 gene (PLCB3) (1) and the VEGF Related Factor (VRF) (2,3). Human PNG does not have any marked similarity to other known genes on the DNA level. PNG showed expression of a 1 kb message in multiple tissues. The predicted protein of 199 amino acids is dominated by glycine, proline and alanine in the N-terminal half and shows similarity to collagen-like proteins. The gene spans approximately 2.5 kb and is divided into 4 exons and 3 introns, and it is located 4.4 kb upstream of the PLCb3 gene, with the 59 ends facing each other. The intergenic region has been completely sequenced, revealing separate CpG islands at both ends of this region. The islands are separated by a stretch of 2 kb, characterized by periodical alteration of the GC content. The 59 flanking region of PNG does not contain TATA or CCAAT boxes which would be in agreement with a house keeping promoter structure (4). To further characterize PNG and to evaluate its status as a MEN1 candidate gene we have cloned and sequenced the murine homologue (png) and investigated its expression pattern in pre- and postnatal mice with in situ hybridization histochemistry. MATERIAL AND METHODS Isolation of cDNAs. Murine png clones were selected from a lambda Zap new born whole brain cDNA library (Stratagene). Primary phages from high density filters (5 × 104 pfu/plate) were identified by hybridization with the 32P-labeled human PNG cDNA (pSOM172). Hybridization and washes of nitrocellulose membranes (Schleicher & Schüll) were carried out at 65°C under stringent conditions. Positive plaques were picked, purified and excised in vivo to produce bacterial colonies containing cDNA clones in pBluescript SK-. Nucleotide sequencing and analysis. Plasmid DNA was mini-prepared through alkaline lysis (5). Plasmid insert DNA was 1
The sequences reported in this paper have been deposited in the EMBL data base (Accession No. X97490). To whom correspondence should be addressed. Fax: +46 8 327734. 3 J.L. and D.K. contributed equally to the work presented here. Abbreviations: PNG, Phospholipase C b3 Neighboring Gene; MEN1, Multiple Endocrine Neoplasia type 1; nt, nucleotide; CNS, central nervous system. 2
335 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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mechanically sheared using a nebulizer (IPI Medical Products, USA no. 4207) (6). DNA fragments between 500–600 bp were blunt-end cloned into pUC18 vector (Pharmacia). Taq Dye Deoxy Terminators kit (ABI) was used for cycle sequencing. The sequences were assembled using the Staden program package (7). Database searches and sequence comparisons were done using FastA and gap programs from Wisconsin Package, Genetics Computer Group, WI, USA. Animals. Timed pregnant (n44) and young adult (n42) mice (C57 inbred strain, ALAB, Sweden) were sacrificed with carbon dioxide, and the relevant tissues were taken out and frozen on a chuck. Tissues were kept at −70°C until further use. Three gestational ages were used in this study; embryonic day 8, (E8), E14 and E17. All animal experiments in this study were approved by the local ethics committee for animal experimentation. In situ hybridization histochemistry. In situ hybridization was performed as previously described (8). Briefly, transverse sections (14 mm) were cut in a cryostat (Microm, Germany), thawed onto Probe-On slides (Fisher Scientific, USA) and stored in black, sealed boxes at −70°C until used. The sequences of the synthetic oligonucleotides complementary to mRNA encoding png were CAGGGGGACT CTGGAAGTAG ACTCGGGGCC CTGTGCTGCC A (complementary to nt 327– 367) and CCCGTCAGTT TAATAACAAT AAAAAACCCC AAAAGTGGAA AACTGAGGG (complementary to nt 855–903). The probes were labeled at the 39-end with deoxyadenosine-alpha[thio]triphosphate[35S] (NEN, USA) using terminal deoxynucleotidyl transferase (IBI, USA) to a specific activity of 7–10 × 108 cpm/mg and hybridized to the sections without pretreatment for 16–18 h at 42°C. The hybridization mixture contained: 50% formamide, 4 × SSC (1 × SSC40.15 M NaCl and 0.015 M sodium-citrate), 1 × Denhardt’s solution (0.02% each of polyvinyl-pyrrolidone, BSA and Ficoll), 1% sarcosyl (N-lauroylsarcosine; Sigma, USA), 0.02 M phosphate buffer (pH 7.0), 10% dextran sulfate (Pharmacia, Sweden), 250 mg/ml yeast tRNA (Sigma), 500 mg/ml sheared and heat denatured salmon sperm DNA (Sigma) and 200 mM dithiothreitol (DTT; LKB, Sweden). In control sections, the specificity of both probes was checked by adding a 20-fold excess of unlabeled probe to the hybridization mixture. In addition adjacent sections were hybridized with probes unrelated to PNG that gave different expression patterns. Following hybridization the sections were washed several times in 1×SSC at 55°C, dehydrated in ethanol and dipped in NTB2 nuclear track emulsion (Kodak, USA) and cover slipped. In some cases, sections were exposed to autoradiographic film (Beta-max autoradiography film Amersham Ltd, UK) prior to emulsion dipping.
RESULTS Mouse png homologues were isolated by screening a murine cDNA library with a human PNG cDNA clone. Five clones of sizes varying from 0.8–1 kb were recovered and sequenced. The cDNA sequences were compiled to give a full length 980 bp cDNA sequence covering the entire open reading frame (609 bp and 39 UTR (275 bp), as well as 96 bp of the 59 UTR. The overall homology of mouse png to human PNG predicted open reading frames were 86% identity and 91% similarity, respectively (fig. 1). The nucleotide sequence were more than 90% identical over the entire coding region. The longest predictable open reading frame of png encodes 203 amino acid residues (fig. 1). The average GC content of the first 300 bp of mouse and human PNG ranges between 80 and 85%, even
FIG. 1. Comparison of predicted amino acid sequences of the human and mouse PNG gene. Amino acid positions are on the left side. 336
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extending into the coding region. From the first 100 predicted amino acids, 65 are alanine, glycine, or proline (25, 25, and 15, respectively). This is also a feature of different types of collagen like genes. Consequently a database search recognized homologies with genes which share this characteristic (not shown). The database search did also identify a close to perfect match with two expressed sequence tags. The first one, H39427, is at rat cDNA clone identified when screening for upregulated genes in astrocytes treated with interferon gamma. The second one, D18307, is a mouse cDNA clone detected when sequencing genes expressed in mouse embryogenesis. In addition a FastA search of the Swissprot protein sequence database, using the predicted protein sequence of png, detected a 28% identity and 74% similarity with the C-terminal region (position 960–1060) of Phospholipase C b2. Protein prediction analysis (9) showed a high probability for a turned structure between amino acid residues 33–44, 74–81 and 89–105. The N- and C-terminal regions are predicted to contain mainly helical structures. Northern blot analysis of RNA from adult mouse tissues (heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis) showed expression in all tissues. The strongest expression was seen in testis, which also had an additional band (fig. 2). This is somewhat different to the pattern observed for human PNG in which similar levels of expression have been identified in all tissues examined (4). The 1 kb murine message presumably corresponds to the shorter of the two testis’ transcripts. The two png oligonucleotide probes gave identical hybridization patterns in all examined tissues. Mouse png expression was detected in the 8 day old embryos (E8), in which positive signals were recorded over structures most likely corresponding to the neuronal tube (not shown). In sagittal sections of the E14 mouse embryo, a positive hybridization signal was present over large parts of the brain, spinal cord, heart, liver, intestines and tongue (fig. 3A). At a later gestational age, E17, the hybridization signal was markedly reduced, except in skeletal muscle, thymus and neocortex
FIG. 2. Autoradiogram of a multiple tissue Northern blot hybridized with mouse png cDNA and GAPDH as a positive control. In all tissues tested a single transcript of 1 kb is detected. In testis an additional band of 2.3 kb is also found. Sizes of RNA markers are given in kb. 337
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FIG. 3. In situ hybridization autoradiographs (A, B) and dark field micrographs (C–F) showing the expression of PNG mRNA in the mouse. Multiple tissues, including the brain (Cx), spinal cord (Sc), heart (Ha), liver (L), intestines (I) and tongue (T), display a positive hybridization signal in the E14 embryo (A). In the E17 embryo (B), PNG is expressed preferably in skeletal muscle (M), neocortex (Cx) and thymus (Th). A strong signal is also recorded over the retina and, to a lesser degree, other parts of the eye in the E17 embryo (C). In the early postnatal mouse (D), a positive signal is present over the thymus (Th), while the heart (Ha) show no or weak labeling. PNG is expressed in the adult mouse testis (E), and grain clusters are shown, in toloudine counterstained sections, over cell body profiles in the tubuli seminiferi (F). Scale bars 4 0.8mm (A), 1.6mm (B), 0.2mm (C), 0.3mm (D), 0.25mm (E), 0.1mm (F). 338
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(fig. 3B). A strong hybridization signal was also recorded over retina (fig. 3C). Most tissues, including the pancreas and the suprarenal, pituitary and thyroid glands, displayed low or no specific labeling in the adult mouse. However, a positive signal was present over the thymus in the early postnatal mouse (fig. 3D) and over adult mouse testis (fig. 3E). In sections counterstained with toluidine, grain clusters were present over cell profiles constituting the tubuli seminiferi (fig. 3F). No specific labeling over background signal was recorded in control sections hybridized with an excess of cold probe (not shown). DISCUSSION Our attempts to characterize genes in the region for Multiple Endocrine Neoplasia type 1 on chromosome 11q13 (10) resulted in the isolation of a number of different transcripts (11). One of them was a 1 kb cDNA clone identified with the cosmid cCLGW4/D11S750, which we previously had isolated from the region (12). The gene was named Phospholipase Neighboring Gene (PNG) due to its close proximity to the Phospholipase C b3 gene (4). Using the human PNG cDNA clone pSOM172 we subsequently isolated and sequenced the mouse homologue of PNG (png). The transcript, 980 bp, of the longest clone has approximately the same size as the transcript determined from Northern blot analysis (fig. 2). The high similarity between the mouse and human PNG (91%) predicted open reading frames suggests that this cDNA clone indeed represents the murine counterpart of the human PNG gene. A region in the N-terminal part of the predicted protein does not display a significant homology with its human analog. Still, on the nucleotide level this region maintains a high homology with human PNG. This discrepancy could be due to an error in one of the two sequences. If so, the quality of the sequences suggests that it is most likely the human one. It is interesting to note that a Swissprot FastA search of the png predicted protein detects a similarity with the Phospholipase C b2 gene, in the region interacting with the Gqa G-protein subunit (13). If this similarity reflects a functional property of PNG it could indicate a regulatory relationship between PNG and the neighboring gene PLCB3 (4). Since MEN1 is generally believed to be a tumor suppressor gene, it seems reasonable to assume that it has normally to be expressed in tissues affected by MEN1 disease, to be considered a candidate gene for this disease. However, it may be that this expression is only seen during specific stages of development. To test the relevance of PNG as a candidate gene for MEN1 we have here investigated its expression during mouse development in general and in endocrine organs in particular. In situ hybridization histochemistry revealed a high png expression in multiple tissues of the midstage mouse embryo, with the highest expression in the developing CNS. Later in prenatal development, png expression was confined preferably to skeletal muscle and the neocortex, while the expression in many of the internal organs and parts of the CNS had been down-regulated. Thus, the expression profile of PNG may be consistent with a functional role during phases of organogenesis characterized by rapid growth. Interestingly, in the postnatal mouse, PNG expression was confined mainly to the thymus and testis, two types of tissue where cell proliferation is very high also in the adult animal. It is also noteworthy that, in the context of testis and thymus expression, thymic carcinoids and infertility have been shown to be involved in cases of MEN1 disease (14, 15). However, we were not able to detect any significant levels of png expression in other organs more commonly affected by MEN1, such as the pituitary. All together, this argues that PNG is not an ideal candidate gene for MEN1. ACKNOWLEDGMENTS This work was supported by grants from the Swedish Cancer Society, the Swedish Medical Research Council, the Magnus Bergvall Foundation, Markus Borgströms Fund, and The Cancer Society in Stockholm. 339
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