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of phosphate from the cytoplasm to the mitochondrial matrix,Chromosomal Localization of the either by proton cotransport or in ion exchange for hydroxylions. Uptake of phosphate into mitochondria is required forMitochondrial Phosphate CarrierATP synthesis during oxidative phosphorylation. PurifiedGene PHC to 12q23 protein from both pig and bovine heart (1, 6) has been recon-stituted into liposomes in an active form. The first completeSharon Marsh,* Nigel P. Carter,* Vincenza Dolce,†amino acid sequence of the PiC has been deduced from bovineVito Iacobazzi,† and Ferdinando Palmieri†,1

heart cDNA (9). The mature protein consists of three tan-*The Sanger Centre, Hinxton Hall, Hinxton, Cambridgeshire, CB10 1RQ, demly repeated sequences of about 100 amino acids, andUnited Kingdom; and †Department of Pharmaco-Biology, Laboratory of these repetitive elements are related to those of the ADP/Biochemistry and Molecular Biology, University of Bari, Bari 70125, Italy ATP carrier, the uncoupling protein, the oxoglutarate carrier,

and the citrate carrier (7), which are well-characterized mem-Received May 8, 1995; accepted July 10, 1995bers of the mitochondrial carrier family. The sequence of thehuman PHC gene has recently been derived from two over-lapping genomic clones, named l12D3 and l2F2, and con-tains evidence of two alternatively spliced forms (4). InitialThe phosphate carrier (PiC) is a ubiquitous protein of thechromosome localization was achieved by PCR amplificationinner mitochondrial membrane that catalyzes the transportof flow-sorted chromosome pools (2) using primers based onthe human cDNA sequence (3) (positions 779–804 for theThe nucleotide sequence of the human PHC gene is deposited withforward primer and positions 1061–1086 for the reversethe GenBank/EMBL Data Bank under Accession No. X77337.primer). Product was generated in the PCR tube containing1 To whom correspondence should be addressed. Telephone: /39

80 5443323. Fax: /39 80 5442770. chromosomes 9, 10, 11, and 12 (see Fig. 1a). In an attempt

FIG. 1. Localization of PHC to chromosome 12q23. (a) PCR of PiC primers on sorted chromosome pools. G, male genomic DNA; N,negative control. (b) PCR of PiC primers on derivative translocation chromosomes. 1, translocation (9; 10)(q33;p11.2); L, der9; R, der10. 2,translocation (9; 10)(q22;q22.1); L, der9; R, der10. 3, translocation (9; 12)(q22;q11); L, der9; R, der12. 4, translocation (9;11)(p24;q11); L,der9; R, der11. (c) FISH of the clone l12D3. The DAPI image has been recolored red for reproduction. (d) Enhanced DAPI-banded image.

GENOMICS 29, 814–815 (1995)0888-7543/95 $12.00Copyright q 1995 by Academic Press, Inc.All rights of reproduction in any form reserved.

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BRIEF REPORTS 815

9. Runswick, M. J., Powell, S. J., Nyren, P., and Walker, J. E.to determine which of these chromosomes carries the gene,(1987). Sequence of the bovine mitochondrial phosphate carrierthe derivative chromosomes from a series of translocationprotein: Structural relationship to ADP/ATP translocase andcell lines (5) involving these four chromosomes as well as athe brown fat mitochondrial uncoupling protein. EMBO J. 6:chromosome 9qh0 sorted from a lymphoblastoid cell line and1367–1373.chromosome 11 sorted from a somatic cell hybrid line were

used as targets for specific PCR. No product was generatedfrom chromosome 11 or 9qh0 or from translocations involv-ing chromosomes 9, 10, and 11. However, with sorted deriva-tive chromosomes from a translocation between chromosomes

Genetic Mapping of a Gene Encoding9 and 12, product was generated from the derivative 9 chro-mosome, which contains chromosome 12q11–qter (see Fig. an Atypical Protein Kinase C, Protein1b). From these results, it was concluded that the phosphate

Kinase C Lambda, to the Proximalcarrier gene was located between 12q11 and 12qter.A more accurate assignment was achieved using fluores- Region of Mouse Chromosome 3

cence in situ hybridization with the clone l12D3, which is afragment of about 15 kb encompassing the entire PHC gene Nandita A. Quaderi,*,1 Fernando Gianfrancesco,†of 7969 bp in length. One microgram of l12D3 DNA was Steve D. M. Brown,* Michele D’Urso,†labeled by nick-translation with biotin–16-dUTP (Boeh-

and Maurizio D’Esposito†ringer). The probe was ethanol precipitated and 80 ng hybrid-ized to normal male metaphase spreads prepared by standard *Department of Biochemistry and Molecular Genetics, St. Mary’smethods. Hybridization and detection methods were essen- Hospital Medical School, Imperial College of Science, Technology andtially as described previously (8), and chromosomes were Medicine, Norfolk Place, London, W2 1PG, United Kingdom; and

†International Institute of Genetics and Biophysics,counterstained with 4,6-diamidino-2-phenylindole (DAPI).CNR, via Marconi, 10, 80125, Naples, ItalyImages were acquired using a Zeiss Axioskop microscope, a

Photometrics KAF 1400 cooled CCD camera, and SmartCap-Received April 27, 1995; accepted July 20, 1995

ture software (Digital Scientific). Chromosomes were identi-fied using enhanced DAPI banding. The clone was localizedto chromosome 12q23 by FISH (see Fig. 1c), confirming theresults from PCR amplification of flow-sorted chromosomes. Protein kinase C are a class of proteins that mediate the

control of cellular responses to external stimuli through thephosphorylation of target proteins (5). These proteins showREFERENCESa molecular heterogeneity by which it is possible to subdividethem into three subclasses (conventional, novel, and atypical)1. Bisaccia, F., and Palmieri, F. (1984). Specific elution from hy-on the basis of their distinct structural and biochemical prop-droxylapatite of the mitochondrial phosphate carrier by cardio-

lipin. Biochim. Biophys. Acta 776: 386–394. erties (6). We have recently described the isolation of a hu-man gene encoding a new member of the atypical class of2. Cotter, F., Naspuri, S., Lam, G., and Young, B. D. (1989). Geneprotein kinase C, protein kinase Ci (PKCi), which we showedmapping by enzymatic amplification from flow-sorted chromo-

somes. Genomics 5: 470–474. mapped to Xq21.3, close to the BTK gene (4). The atypicalclass of protein kinase C contains at least three members,3. Dolce, V., Fiermonte, G., Messina, A., and Palmieri, F. (1991).

Nucleotide sequence of a human heart cDNA encoding the mito- protein kinase Cl, protein kinase Cz, and the previously men-chondrial phosphate carrier. DNA Sequence—J. Sequencing tioned protein kinase Ci, although further members haveand Mapping 2: 133–135. been predicted to exist on the basis of genomic Southern blot-

4. Dolce, V., Iacobazzi, V., Palmieri, F., and Walker, J. E. (1994). ting (7). To clone the mouse genes for new members of theThe sequence of human and bovine genes of the phosphate car- atypical class of protein kinase C, we screened a mouse 11.5-rier from mitochondria contain evidence of alternatively spliced day embryonic cDNA library with a 2.2-kb cDNA represent-forms. J. Biol. Chem. 269: 10451–10460. ing the entire human PKCi gene. Eight cDNA clones were

5. Goudie, D. R., Yuille, M. A., Leversha, M. A., Furlong, R. A., isolated, of which six were either partially or fully sequenced.Carter, N. P., Lush, M. J., Affara, N. A., and Ferguson-Smith, Their sizes ranged from 2.7 to 2.9 kb and appeared to beM. A. (1993). Multiple self-healing squamous epitheliomata overlapping fragments of the same transcript. A FASTA(ESS1) mapped to chromosome 9q22–q31 in families with com-

search of the GenBank database using 1.5 kb of consensusmon ancestry. Nature Genet. 3: 165–169.cDNA sequence, containing mainly 3 * untranslated se-

6. Kolbe, H. V. J., Costello, D., Wong, A., Lu, R. C., and Wohlrab, quence, revealed 99% identity at the nucleotide level withH. (1984). Mitochondrial phosphate transport. J. Biol. Chem.mouse protein kinase Cl (Accession No. D28577) (1). Protein259: 9115–9120.kinase Cl (PKCl) has been previously characterized by Aki-

7. Palmieri, F. (1994). Mitochondrial carrier proteins. FEBS Lett.moto et al. (1), but the gene encoding it had not been mapped346: 48–54.in mouse or human. Mouse PKCl is expressed abundantly in

8. Pinkel, D., Landegant, J., Collins, C., Fuscoe, J., Segraves, R.,Lucas, J., and Gray, J. (1988). Fluorescence in situ hybridiza-tion with human chromosome-specific libraries: Detection of tri- 1 To whom correspondence should be addressed. Telephone: /44

171 723 1254, x5495. Fax: /44 171 706 3272. E-mail: naq30@sm.ic.somy 21 and translocations of chromosome 4. Proc. Natl. Acad.Sci. USA 85: 9138–9142. ac.uk.

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