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Genetic Mapping of a Gene Encoding an Atypical Protein Kinase C, Protein Kinase C Lambda, to the Proximal Region of Mouse Chromosome 3
815
BRIEF REPORTS
to determine which of these chromosomes carries the gene, the derivative chromosomes from a series of translocation cell lines (5) involving these four chromosomes as well as a chromosome 9qh0 sorted from a lymphoblastoid cell line and chromosome 11 sorted from a somatic cell hybrid line were used as targets for specific PCR. No product was generated from chromosome 11 or 9qh0 or from translocations involving chromosomes 9, 10, and 11. However, with sorted derivative chromosomes from a translocation between chromosomes 9 and 12, product was generated from the derivative 9 chromosome, which contains chromosome 12q11 – qter (see Fig. 1b). From these results, it was concluded that the phosphate carrier gene was located between 12q11 and 12qter. A more accurate assignment was achieved using fluorescence in situ hybridization with the clone l12D3, which is a fragment of about 15 kb encompassing the entire PHC gene of 7969 bp in length. One microgram of l12D3 DNA was labeled by nick-translation with biotin –16-dUTP (Boehringer). The probe was ethanol precipitated and 80 ng hybridized to normal male metaphase spreads prepared by standard methods. Hybridization and detection methods were essentially as described previously (8), and chromosomes were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Images were acquired using a Zeiss Axioskop microscope, a Photometrics KAF 1400 cooled CCD camera, and SmartCapture software (Digital Scientific). Chromosomes were identified using enhanced DAPI banding. The clone was localized to chromosome 12q23 by FISH (see Fig. 1c), confirming the results from PCR amplification of flow-sorted chromosomes. REFERENCES 1. Bisaccia, F., and Palmieri, F. (1984). Specific elution from hydroxylapatite of the mitochondrial phosphate carrier by cardiolipin. Biochim. Biophys. Acta 776: 386– 394. 2. Cotter, F., Naspuri, S., Lam, G., and Young, B. D. (1989). Gene mapping by enzymatic amplification from flow-sorted chromosomes. Genomics 5: 470– 474. 3. Dolce, V., Fiermonte, G., Messina, A., and Palmieri, F. (1991). Nucleotide sequence of a human heart cDNA encoding the mitochondrial phosphate carrier. DNA Sequence — J. Sequencing and Mapping 2: 133– 135. 4. Dolce, V., Iacobazzi, V., Palmieri, F., and Walker, J. E. (1994). The sequence of human and bovine genes of the phosphate carrier from mitochondria contain evidence of alternatively spliced forms. J. Biol. Chem. 269: 10451 –10460. 5. Goudie, D. R., Yuille, M. A., Leversha, M. A., Furlong, R. A., Carter, N. P., Lush, M. J., Affara, N. A., and Ferguson-Smith, M. A. (1993). Multiple self-healing squamous epitheliomata (ESS1) mapped to chromosome 9q22 – q31 in families with common ancestry. Nature Genet. 3: 165– 169. 6. Kolbe, H. V. J., Costello, D., Wong, A., Lu, R. C., and Wohlrab, H. (1984). Mitochondrial phosphate transport. J. Biol. Chem. 259: 9115 –9120. 7. Palmieri, F. (1994). Mitochondrial carrier proteins. FEBS Lett. 346: 48– 54. 8. Pinkel, D., Landegant, J., Collins, C., Fuscoe, J., Segraves, R., Lucas, J., and Gray, J. (1988). Fluorescence in situ hybridization with human chromosome-specific libraries: Detection of trisomy 21 and translocations of chromosome 4. Proc. Natl. Acad. Sci. USA 85: 9138 –9142.
9. Runswick, M. J., Powell, S. J., Nyren, P., and Walker, J. E. (1987). Sequence of the bovine mitochondrial phosphate carrier protein: Structural relationship to ADP/ATP translocase and the brown fat mitochondrial uncoupling protein. EMBO J. 6: 1367 –1373.
Genetic Mapping of a Gene Encoding an Atypical Protein Kinase C, Protein Kinase C Lambda, to the Proximal Region of Mouse Chromosome 3 Nandita A. Quaderi,*,1 Fernando Gianfrancesco,† Steve D. M. Brown,* Michele D’Urso,† and Maurizio D’Esposito† *Department of Biochemistry and Molecular Genetics, St. Mary’s Hospital Medical School, Imperial College of Science, Technology and Medicine, Norfolk Place, London, W2 1PG, United Kingdom; and †International Institute of Genetics and Biophysics, CNR, via Marconi, 10, 80125, Naples, Italy Received April 27, 1995; accepted July 20, 1995
Protein kinase C are a class of proteins that mediate the control of cellular responses to external stimuli through the phosphorylation of target proteins (5). These proteins show a molecular heterogeneity by which it is possible to subdivide them into three subclasses (conventional, novel, and atypical) on the basis of their distinct structural and biochemical properties (6). We have recently described the isolation of a human gene encoding a new member of the atypical class of protein kinase C, protein kinase Ci (PKCi), which we showed mapped to Xq21.3, close to the BTK gene (4). The atypical class of protein kinase C contains at least three members, protein kinase Cl, protein kinase Cz, and the previously mentioned protein kinase Ci, although further members have been predicted to exist on the basis of genomic Southern blotting (7). To clone the mouse genes for new members of the atypical class of protein kinase C, we screened a mouse 11.5day embryonic cDNA library with a 2.2-kb cDNA representing the entire human PKCi gene. Eight cDNA clones were isolated, of which six were either partially or fully sequenced. Their sizes ranged from 2.7 to 2.9 kb and appeared to be overlapping fragments of the same transcript. A FASTA search of the GenBank database using 1.5 kb of consensus cDNA sequence, containing mainly 3 * untranslated sequence, revealed 99% identity at the nucleotide level with mouse protein kinase Cl (Accession No. D28577) (1). Protein kinase Cl (PKCl) has been previously characterized by Akimoto et al. (1), but the gene encoding it had not been mapped in mouse or human. Mouse PKCl is expressed abundantly in 1 To whom correspondence should be addressed. Telephone: /44 171 723 1254, x5495. Fax: /44 171 706 3272. E-mail: [email protected]. ac.uk.
GENOMICS
29, 815–816 (1995)
0888-7543/95 $12.00 Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
/ m4055$brep
10-03-95 15:58:40
gnmbr
AP-Genomics
816
BRIEF REPORTS
FIG. 1. (A) Segregation of a Bgl II restriction fragment length variant at the Pkcl locus in backcross progeny from a C57BL/6-Mus spretus interspecific backcross (2). A 330-bp EcoRI/Sal I Pkcl probe containing mostly 3* untranslated sequence detects a C57BL/6 parental allele of 7 kb (track a) and a Mus spretus parental allele of 2.9 kb (track b). Following Bgl II digestion, approximately 2 mg of DNA from the backcross progeny mice were fractionated on 1% agarose gels and transferred to Hybond-N/ nylon membranes (Amersham International). Probes were labeled by random oligo priming to a specific activity of 109 dpm/mg. Filters were hybridized overnight at 657C in 61 SSC, 0.5% SDS, 10% (w/v) dextran sulfate, in the presence of 100 mg/ml sheared salmon sperm DNA and 50 ng labeled probe. Filters were washed in 31 SSC, 0.1% SDS at 657C and subsequently autoradiographed with Kodak X-ray film at 0707C with intensification for 24– 72 h. (B) Haplotype analysis of 59 backcross progeny demonstrating linkage of Pkcl to the interval between D3Mit61 and D3Nds27 on mouse chromosome 3. Genetic order was determined by minimizing the number of observed recombinants. Intergenic distances with standard errors are given.
undifferentiated P19 mouse embryonal carcinoma cells and shows a decrease in expression with differentiation. It is known to be expressed in a wide variety of tissues, but its precise biological function has yet to be elucidated (1). To determine a genetic map position for the PKCl gene (Pkcl), we utilized the EUCIB backcross, which has been previously described (2). Briefly, 982 progeny were produced by an interspecific backcross between C57BL/6 and Mus spretus and scored for at least 3 anchor loci on each chromosome. A 330-bp EcoRI/SalI Pkcl probe containing mostly 3 * untranslated sequence was generated and used in all subsequent experiments. The Pkcl probe detected a Bgl II restriction fragment length variation between C57BL/6 and M. spretus genomic DNAs (Fig. 1A). The Pkcl probe detected a C57BL/6 parental allele of 7 kb and a M. spretus parental allele of 2.9 kb. DNAs from 59 randomly chosen progeny mice from the EUCIB backcross were digested with BglII and hybridized with Pkcl. The Pkcl probe showed linkage to the anchor markers D3Nds27 and D3Mit61 on chromosome 3 (lod scores of 7.69 and 6.25, respectively). The results of the haplotype analysis are summarized in Fig. 1B. Of the 59 mice analyzed, 7/56 showed recombination events between Pkcl and D3Nds27 and 7/53 showed recombination events between Pkcl and D3Mit61. Minimizing the number of recombinants observed among the panel of 59 mice scored indicated that Pkcl maps distal to D3Mit61 and proximal to D3Nds27 (Fig. 1B). The locus D3Nds27 is derived from the Il2 gene, which is assigned a genetic map position of 18.5 cM on the chromosome 3 committee linkage map (8). The mouse Pkcl gene has been genetically mapped to the proximal region of chromosome 3 between D3Mit61 and D3Nds27. This region has previously been shown to be homologous with human 8q13 –q22, 4q26 –q27, and 3q24 –q28 (8). Several uncloned mouse mutations have been reported to map to this area; however, further investigations to clarify the biological significance of PKCl are needed to determine whether Pkcl is a candidate gene for any of these. To date, the genes for only two other protein kinase C have been mapped in either human or mouse: the gene for protein kinase Ci has been mapped to Xq21.3 in human (4), and the gene for protein kinase Ch has been mapped to proximal
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chromosome 12 in mouse (3). Neither of these genes has been associated with an inherited phenotype. ACKNOWLEDGMENTS The authors thank Dr. A. Ciccodicola for his help during the initial part of the work and Mrs. M. Terracciano for her excellent technical assistance. This work was supported in part by P. F. ‘‘Ingegneria Genetica’’ and from Telethon Grant 417 to M. D’Urso and Medical Research Council Grant G9201373 to SDMB.
REFERENCES 1. Akimoto, K., Mizuno, K., Osada, S., Hirai, S., Tanuma, S., Suzuki, K., and Ohno, S. (1994). A new member of the third class in the protein kinase C family, PKC lambda, expressed dominantly in undifferentiated mouse embyronal carcinoma cell line and also in many tissues and cells. J. Biol. Chem. 269: 12677 – 12683. 2. Breen, M., and the European Backcross Collaborative Group (1994). Towards high resolution maps of the mouse and human genomes— A facility for ordering markers to 0.1 cM resolution. Hum. Mol. Genet. 3: 621–627. 3. Canzian, F., Gariboldi, M., Manenti, G., De Gregorio, L., Osada, S., Ohno, S., Dragani, T. A., and Pierotti, M. A. (1994). Expression in lung tumours and genetic mapping of the novel murine protein kinase C eta. Mol. Carcinogen 9: 111– 113. 4. Mazzarella, R., Ciccodicola, A., Esposito, T., Arcucci, A., Migliaccio, C., Jones, C., Schlessinger, D., D’Urso, M., and D’Esposito, M. (1995). A human protein kinase C iota gene is closely linked to the BTK gene in Xq21.3. Genomics 26: 629–631. 5. Nishizuka, Y. (1988). The molecular heterogeneity of protein kinase C and its implication for cellular regulation. Nature 334: 661– 665. 6. Nishizuka, Y. (1992). Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258: 607– 614. 7. Ono, Y., Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K., and Nishizuka, Y. (1989). Protein kinase C zeta subspecies from rat brain: Its structure, expression, and properties. Proc. Natl. Acad. Sci. USA 86: 3099 –3103. 8. Prins, J.-B., Meisler, M. H., and Seldin, M. F. (1994). Mouse chromosome 3. Mamm. Genome 5: S40 –S50.
AP-Genomics
BRIEF REPORTS
to determine which of these chromosomes carries the gene, the derivative chromosomes from a series of translocation cell lines (5) involving these four chromosomes as well as a chromosome 9qh0 sorted from a lymphoblastoid cell line and chromosome 11 sorted from a somatic cell hybrid line were used as targets for specific PCR. No product was generated from chromosome 11 or 9qh0 or from translocations involving chromosomes 9, 10, and 11. However, with sorted derivative chromosomes from a translocation between chromosomes 9 and 12, product was generated from the derivative 9 chromosome, which contains chromosome 12q11 – qter (see Fig. 1b). From these results, it was concluded that the phosphate carrier gene was located between 12q11 and 12qter. A more accurate assignment was achieved using fluorescence in situ hybridization with the clone l12D3, which is a fragment of about 15 kb encompassing the entire PHC gene of 7969 bp in length. One microgram of l12D3 DNA was labeled by nick-translation with biotin –16-dUTP (Boehringer). The probe was ethanol precipitated and 80 ng hybridized to normal male metaphase spreads prepared by standard methods. Hybridization and detection methods were essentially as described previously (8), and chromosomes were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Images were acquired using a Zeiss Axioskop microscope, a Photometrics KAF 1400 cooled CCD camera, and SmartCapture software (Digital Scientific). Chromosomes were identified using enhanced DAPI banding. The clone was localized to chromosome 12q23 by FISH (see Fig. 1c), confirming the results from PCR amplification of flow-sorted chromosomes. REFERENCES 1. Bisaccia, F., and Palmieri, F. (1984). Specific elution from hydroxylapatite of the mitochondrial phosphate carrier by cardiolipin. Biochim. Biophys. Acta 776: 386– 394. 2. Cotter, F., Naspuri, S., Lam, G., and Young, B. D. (1989). Gene mapping by enzymatic amplification from flow-sorted chromosomes. Genomics 5: 470– 474. 3. Dolce, V., Fiermonte, G., Messina, A., and Palmieri, F. (1991). Nucleotide sequence of a human heart cDNA encoding the mitochondrial phosphate carrier. DNA Sequence — J. Sequencing and Mapping 2: 133– 135. 4. Dolce, V., Iacobazzi, V., Palmieri, F., and Walker, J. E. (1994). The sequence of human and bovine genes of the phosphate carrier from mitochondria contain evidence of alternatively spliced forms. J. Biol. Chem. 269: 10451 –10460. 5. Goudie, D. R., Yuille, M. A., Leversha, M. A., Furlong, R. A., Carter, N. P., Lush, M. J., Affara, N. A., and Ferguson-Smith, M. A. (1993). Multiple self-healing squamous epitheliomata (ESS1) mapped to chromosome 9q22 – q31 in families with common ancestry. Nature Genet. 3: 165– 169. 6. Kolbe, H. V. J., Costello, D., Wong, A., Lu, R. C., and Wohlrab, H. (1984). Mitochondrial phosphate transport. J. Biol. Chem. 259: 9115 –9120. 7. Palmieri, F. (1994). Mitochondrial carrier proteins. FEBS Lett. 346: 48– 54. 8. Pinkel, D., Landegant, J., Collins, C., Fuscoe, J., Segraves, R., Lucas, J., and Gray, J. (1988). Fluorescence in situ hybridization with human chromosome-specific libraries: Detection of trisomy 21 and translocations of chromosome 4. Proc. Natl. Acad. Sci. USA 85: 9138 –9142.
9. Runswick, M. J., Powell, S. J., Nyren, P., and Walker, J. E. (1987). Sequence of the bovine mitochondrial phosphate carrier protein: Structural relationship to ADP/ATP translocase and the brown fat mitochondrial uncoupling protein. EMBO J. 6: 1367 –1373.
Genetic Mapping of a Gene Encoding an Atypical Protein Kinase C, Protein Kinase C Lambda, to the Proximal Region of Mouse Chromosome 3 Nandita A. Quaderi,*,1 Fernando Gianfrancesco,† Steve D. M. Brown,* Michele D’Urso,† and Maurizio D’Esposito† *Department of Biochemistry and Molecular Genetics, St. Mary’s Hospital Medical School, Imperial College of Science, Technology and Medicine, Norfolk Place, London, W2 1PG, United Kingdom; and †International Institute of Genetics and Biophysics, CNR, via Marconi, 10, 80125, Naples, Italy Received April 27, 1995; accepted July 20, 1995
Protein kinase C are a class of proteins that mediate the control of cellular responses to external stimuli through the phosphorylation of target proteins (5). These proteins show a molecular heterogeneity by which it is possible to subdivide them into three subclasses (conventional, novel, and atypical) on the basis of their distinct structural and biochemical properties (6). We have recently described the isolation of a human gene encoding a new member of the atypical class of protein kinase C, protein kinase Ci (PKCi), which we showed mapped to Xq21.3, close to the BTK gene (4). The atypical class of protein kinase C contains at least three members, protein kinase Cl, protein kinase Cz, and the previously mentioned protein kinase Ci, although further members have been predicted to exist on the basis of genomic Southern blotting (7). To clone the mouse genes for new members of the atypical class of protein kinase C, we screened a mouse 11.5day embryonic cDNA library with a 2.2-kb cDNA representing the entire human PKCi gene. Eight cDNA clones were isolated, of which six were either partially or fully sequenced. Their sizes ranged from 2.7 to 2.9 kb and appeared to be overlapping fragments of the same transcript. A FASTA search of the GenBank database using 1.5 kb of consensus cDNA sequence, containing mainly 3 * untranslated sequence, revealed 99% identity at the nucleotide level with mouse protein kinase Cl (Accession No. D28577) (1). Protein kinase Cl (PKCl) has been previously characterized by Akimoto et al. (1), but the gene encoding it had not been mapped in mouse or human. Mouse PKCl is expressed abundantly in 1 To whom correspondence should be addressed. Telephone: /44 171 723 1254, x5495. Fax: /44 171 706 3272. E-mail: [email protected]. ac.uk.
GENOMICS
29, 815–816 (1995)
0888-7543/95 $12.00 Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
/ m4055$brep
10-03-95 15:58:40
gnmbr
AP-Genomics
816
BRIEF REPORTS
FIG. 1. (A) Segregation of a Bgl II restriction fragment length variant at the Pkcl locus in backcross progeny from a C57BL/6-Mus spretus interspecific backcross (2). A 330-bp EcoRI/Sal I Pkcl probe containing mostly 3* untranslated sequence detects a C57BL/6 parental allele of 7 kb (track a) and a Mus spretus parental allele of 2.9 kb (track b). Following Bgl II digestion, approximately 2 mg of DNA from the backcross progeny mice were fractionated on 1% agarose gels and transferred to Hybond-N/ nylon membranes (Amersham International). Probes were labeled by random oligo priming to a specific activity of 109 dpm/mg. Filters were hybridized overnight at 657C in 61 SSC, 0.5% SDS, 10% (w/v) dextran sulfate, in the presence of 100 mg/ml sheared salmon sperm DNA and 50 ng labeled probe. Filters were washed in 31 SSC, 0.1% SDS at 657C and subsequently autoradiographed with Kodak X-ray film at 0707C with intensification for 24– 72 h. (B) Haplotype analysis of 59 backcross progeny demonstrating linkage of Pkcl to the interval between D3Mit61 and D3Nds27 on mouse chromosome 3. Genetic order was determined by minimizing the number of observed recombinants. Intergenic distances with standard errors are given.
undifferentiated P19 mouse embryonal carcinoma cells and shows a decrease in expression with differentiation. It is known to be expressed in a wide variety of tissues, but its precise biological function has yet to be elucidated (1). To determine a genetic map position for the PKCl gene (Pkcl), we utilized the EUCIB backcross, which has been previously described (2). Briefly, 982 progeny were produced by an interspecific backcross between C57BL/6 and Mus spretus and scored for at least 3 anchor loci on each chromosome. A 330-bp EcoRI/SalI Pkcl probe containing mostly 3 * untranslated sequence was generated and used in all subsequent experiments. The Pkcl probe detected a Bgl II restriction fragment length variation between C57BL/6 and M. spretus genomic DNAs (Fig. 1A). The Pkcl probe detected a C57BL/6 parental allele of 7 kb and a M. spretus parental allele of 2.9 kb. DNAs from 59 randomly chosen progeny mice from the EUCIB backcross were digested with BglII and hybridized with Pkcl. The Pkcl probe showed linkage to the anchor markers D3Nds27 and D3Mit61 on chromosome 3 (lod scores of 7.69 and 6.25, respectively). The results of the haplotype analysis are summarized in Fig. 1B. Of the 59 mice analyzed, 7/56 showed recombination events between Pkcl and D3Nds27 and 7/53 showed recombination events between Pkcl and D3Mit61. Minimizing the number of recombinants observed among the panel of 59 mice scored indicated that Pkcl maps distal to D3Mit61 and proximal to D3Nds27 (Fig. 1B). The locus D3Nds27 is derived from the Il2 gene, which is assigned a genetic map position of 18.5 cM on the chromosome 3 committee linkage map (8). The mouse Pkcl gene has been genetically mapped to the proximal region of chromosome 3 between D3Mit61 and D3Nds27. This region has previously been shown to be homologous with human 8q13 –q22, 4q26 –q27, and 3q24 –q28 (8). Several uncloned mouse mutations have been reported to map to this area; however, further investigations to clarify the biological significance of PKCl are needed to determine whether Pkcl is a candidate gene for any of these. To date, the genes for only two other protein kinase C have been mapped in either human or mouse: the gene for protein kinase Ci has been mapped to Xq21.3 in human (4), and the gene for protein kinase Ch has been mapped to proximal
/ m4055$brep
10-03-95 15:58:40
gnmbr
chromosome 12 in mouse (3). Neither of these genes has been associated with an inherited phenotype. ACKNOWLEDGMENTS The authors thank Dr. A. Ciccodicola for his help during the initial part of the work and Mrs. M. Terracciano for her excellent technical assistance. This work was supported in part by P. F. ‘‘Ingegneria Genetica’’ and from Telethon Grant 417 to M. D’Urso and Medical Research Council Grant G9201373 to SDMB.
REFERENCES 1. Akimoto, K., Mizuno, K., Osada, S., Hirai, S., Tanuma, S., Suzuki, K., and Ohno, S. (1994). A new member of the third class in the protein kinase C family, PKC lambda, expressed dominantly in undifferentiated mouse embyronal carcinoma cell line and also in many tissues and cells. J. Biol. Chem. 269: 12677 – 12683. 2. Breen, M., and the European Backcross Collaborative Group (1994). Towards high resolution maps of the mouse and human genomes— A facility for ordering markers to 0.1 cM resolution. Hum. Mol. Genet. 3: 621–627. 3. Canzian, F., Gariboldi, M., Manenti, G., De Gregorio, L., Osada, S., Ohno, S., Dragani, T. A., and Pierotti, M. A. (1994). Expression in lung tumours and genetic mapping of the novel murine protein kinase C eta. Mol. Carcinogen 9: 111– 113. 4. Mazzarella, R., Ciccodicola, A., Esposito, T., Arcucci, A., Migliaccio, C., Jones, C., Schlessinger, D., D’Urso, M., and D’Esposito, M. (1995). A human protein kinase C iota gene is closely linked to the BTK gene in Xq21.3. Genomics 26: 629–631. 5. Nishizuka, Y. (1988). The molecular heterogeneity of protein kinase C and its implication for cellular regulation. Nature 334: 661– 665. 6. Nishizuka, Y. (1992). Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258: 607– 614. 7. Ono, Y., Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K., and Nishizuka, Y. (1989). Protein kinase C zeta subspecies from rat brain: Its structure, expression, and properties. Proc. Natl. Acad. Sci. USA 86: 3099 –3103. 8. Prins, J.-B., Meisler, M. H., and Seldin, M. F. (1994). Mouse chromosome 3. Mamm. Genome 5: S40 –S50.
AP-Genomics