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P deficiency in close family members, cases of nonfamilial Cloning and Molecular Characterizationaminopeptidase P deficiency, and normal subjects.of a Cross-Homologous Zinc

REFERENCES Finger Locus ZNF2041. Bazan, J. F., Weaver, L. H., Roderick, S. L., Huber, R., and Mat- Jason Partridge,* Daniel F. Wallace,*

thews, B. W. (1994). Sequence and structure comparison sug- Alexis Robertson,* Margaret F. Fox,†gest that methionine aminopeptidase, prolidase, aminopepti-

J. Paul Simons,‡ James S. Dooley,*dase P, and creatinase share a common fold. Proc. Natl. Acad.and Ann P. Walker*,1

Sci. USA 91: 2473–2477.2. Blau, N., Niederwieser, A., and Shmerling, D. H. (1988). Pepti-

*Joint Department of Medicine, University College London Medical Schoolduria presumably caused by aminopeptidase-P deficiency—Aand Royal Free Hospital School of Medicine (Royal Free Hospital Campus),new inborn error of metabolism. J. Inherit. Metab. Dis. 11: 240– Pond Street, London, NW3 2QG, United Kingdom; †MRC Human

242. Biochemical Genetics Unit, Galton Laboratory, Wolfson House,3. Chen, X., Orphanos, S. E., Ryan, J. W., Chung, A. Y. K., Hess, 4 Stephenson Way, London, NW1 2HE, United Kingdom; and

D. C., and Catravas, J. D. (1991). Species variation in pulmo- ‡Department of Anatomy and Developmental Biology,nary endothelial aminopeptidase P activity. J. Pharmacol. Expt. Royal Free Hospital School of Medicine, Rowland Hill

Street, London, NW3 2PF, United KingdomTher. 259: 1301–1307.4. Harbeck, H. T., and Mentlein, R. (1991). Aminopeptidase P from

Received January 29, 1998; accepted March 19, 1998rat brain—Purification and action on bioactive peptides. Eur.J. Biochem. 198: 451–458.

5. Hooper, N. M., Hryszko, J., and Turner, A. J. (1990). Purifica-tion and characterization of pig kidney aminopeptidase P. Bio-

As part of a search for genes from the hemochromatosischem. J. 267: 509–515.region, we isolated novel transcripts from chromosome 6p21.36. Medeiros, M. D. S., and Turner, A. J. (1994). Processing andby direct hybridization of P1-derived artificial chromosomesmetabolism of peptide-YY: Pivotal roles of dipeptidylpeptidase-(PACs) from around D6S1260 (5) (Fig. 1a) to a small intestineIV, aminopeptidase P, and endopeptidase-24.11. Endocrinology

134: 2088–2094. cDNA library (Clontech). Four cDNAs corresponded to a sin-gle locus encompassing a 1.1-kb reading frame (Fig. 1b). The7. Mentlein, R., von Kolszynshi, M., Sprang, R., and Lucius, R.

(1990). Proline-specific proteases in cultivated neuronal and cDNA and genomic sequences were identical, confirming theglial cells. Brain Res. 527: 159–162. mapping.

The ZNF204 transcript contained a total of 18 two-cys-8. Orawski, A. T., and Simmons, W. H. (1995). Purification andproperties of membrane-bound aminopeptidase P from rat lung. teine–two-histidine (C2H2) zinc finger motifs and the primerBiochemistry 34: 11227–11236. sequences for two expressed sequence tags (ESTs), STSG-

9. Revann, T. R., Ryan, J. W., Berryer, P., and Valido, F. (1991). 9945 (http://www-genome.wi.mit.edu) and EST449 (4). As ex-Aminopeptidase P is disposed on guinea pig vascular endothe- pected, STSG-9945 was located within the 3 * untranslatedlium and some epithelia. FASEB J. 5: A1579. [Abstract] region (UTR), originating from oligo(dT) priming of the

10. Rusu, I., and Yaron, A. (1992). Aminopeptidase P from human poly(A) tail. However, EST449 was derived from an internalleukocytes. Eur. J. Biochem. 210: 93–100. poly(A) region close to the 5* end of the transcript (Fig. 1b).

11. Ryan, J. W. (1989). Peptidase enzymes of the pulmonary vascu- The complete sequence of the ZNF204 cDNAs revealed thelar surface. Am. J. Physiol. 257: L53–L60. presence of a premature stop codon upstream of zinc finger

12. Ryan, J. W., Berryer, P., Chung, A. Y. K., and Sheffy, D. H., Jr., motifs 14–18 and a frameshift mutation within zinc finger(1994). Characterization of rat pulmonary vascular aminopepti- motif 17 (Fig. 1c). Sequence analysis of both genomic anddase P in vivo: Role in the inactivation of bradykinin. J. Phar- cDNA clones revealed that 10 of the 18 zinc finger motifsmacol. Exp. Ther. 269: 941–947. conformed fully with the consensus (C-X2,4-C-X3-F-X5-L-X2-

13. Ryan, J. W., Chung, A. Y. K., Berryer, P., and Sheffy, D. H., Jr., H-X3,4-H). The remaining 8 motifs contain mutations that(1992). A radioassay for aminoacylproline hydrolase (aminopep- predict amino acid substitutions at conserved cysteine andtidase P) activity. Biochim. Biophys. Acta, 1119: 133–139. histidine residues. Mutations of these conserved residues

14. Ryan, J. W., Valido, F., Berryer, P., Chung, A. Y. K., and Ripka, may eliminate the function of zinc finger proteins (2). Also,J. E. (1992). Purification and characterization of guinea pig se- amplification of the genomic ZNF204 locus in three overlap-rum aminoacylproline hydrolase (aminopeptidase P). Biochim. ping segments using cDNA primers (Fig. 1c) revealed thatBiophys. Acta, 1119: 140–147.

the 2.7-kb ZNF204 locus contains no introns. Together, these15. Vanhoof, G., DeBlock, J., De Meester, I., Scharpe, S., and De data indicate that this locus is most likely an expressed pro-

Potter, W. P. (1992). Localization and characterization ofcessed pseudogene. Consistent with this cDNA being non-aminopeptidase P in bovine adrenal medulla. Neurochem. Int.functional is the presence of multiple potential start and in-21: 203–208.frame termination codons within the putative 5* UTR.16. Venema, R. C., Ju, H., Zou, R., Venema, V. J., and Ryan, J. W.

To map ZNF204 and search for a ‘‘parent’’ locus, a somatic(1997). Cloning and tissue distribution of human membrane-bound aminopeptidase P. Biochim. Biophys. Acta. 1354: 45–48.

17. Yaron, A., and Naider, F. (1993). Proline-dependent structural 1 To whom correspondence should be addressed. Telephone: 44 171794 0500, ext. 3986/3949. Fax: 44 171 830 2631. E-mail: a_walker@and biological properties of peptides and proteins. CRC Crit.

Rev. Biochem. Mol. Biol. 28: 31–81. rfhsm.ac.uk.

GENOMICS 50, 116–118 (1998)ARTICLE NO. GE985318

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FIG. 1. Mapping, isolation, and characterization of ZNF204 (GenBank Accession No. AF033199). (a) Fine-resolution genomic maparound D6S1260. PAC clones used to screen the small intestinal cDNA library are shown; dots indicate the presence of markers (4, 5, 8, 9).(b) Overlapping cDNA clones showing the position of EST449 and STSG-9945; a double slash indicates the junction of ZNF204 cDNA cloneswith chimeric sequence. (c) Schematic representation of ZNF204 showing the 1.1-kb open reading frame consensus sequence and thepositions of its start ATG (arrow at 5* end) and polyadenylation consensus sequence (pA). Blocks indicate the positions of individual zincfinger motifs with asterisks representing termination codons and a crooked arrow indicating the location of the frameshift mutation. Theposition of the PCR hybridization probe is indicated above the consensus. Below the consensus, the three bars represent the three overlappingPCR products amplified from genomic DNA using cDNA primers; cDNA and genomic DNA products were of identical size. (d) ZNF204cross-hybridizes with multiple zinc finger loci. Southern blot of HindIII-digested DNA (10 mg) from pig, cow, hamster, and human probedwith an internal PCR fragment from ZNF204 (c). Southern blots were rinsed once with 41 SSC at room temperature, twice for 20 min in0.51 SSC, 0.1% SDS at 427C, and once for 20 min in 0.51 SSC, 0.1% SDS at 657C. A ladder of bands increasing uniformly by approximately80 bp was seen in all lanes reflecting cross-hybridization of ZNF204 to multiple zinc finger loci across species.

cell hybrid panel was screened by PCR using three sets of from coding sequence. Taking the mapping and homology anal-yses together, it seems likely that the putative parent locus isprimers (Fig. 1c); however, additional chromosomal loci were

not detected. Non-zinc-finger portions of the locus (EST449, in 6p21.3 and possibly related to ZNF184. Indeed, ZNF204and ZNF184 both hybridize to a 3.2-kb transcript on NorthernSTSG-9945; Fig. 1b) gave only a smear when hybridized to

genomic DNA, limiting their use in the search for a parent analysis (data not shown and Ref. 3), consistent with a sharedancestry. The processed appearance of ZNF204 suggests alocus. Fluorescence in situ hybridization (FISH) analysis

with PAC pCYPAC2N11g1 revealed a strong signal only on retrotransposed intermediate. Localized enrichment of retro-transposed elements has not been previously demonstrated.chromosome 6p21.3. With cDNA3 (2.7 kb), a small specific

signal was seen only in 6p21.3. Of 11 cells imaged, 14/44 Interestingly, the first 90 bp of cDNA sequence has homologywith the THE1c repeat element family. Further studies would(32%) of chromatids contained the signal. Chromosome

6p21.3 contains a cluster of at least six other C2H2 zinc finger be required to investigate the potential involvement of thissequence in a mechanism of local retrotransposition.genes (ZNF165, ZNF193, ZNF192, ZNF184, SRE-ZBP, and

WI-7080), suggesting the possibility that the parent locus To analyze the evolutionary conservation of ZNF204, a zooblot of HindIII-digested DNA was screened using an internalmay also be in 6p21.3.

To investigate the similarity of ZNF204 to published se- PCR fragment probe (Fig. 1c). This showed a ladder of hy-bridizing bands increasing uniformly in size by approxi-quences, homology analysis was performed using the BLAST

program (1). Reanalysis of the highest 10 BLAST homologies mately 80 bp in all species tested (pig, cow, hamster, andhuman; Fig. 1d). This step-wise increase in the size of frag-using the BESTFIT (GCG) program showed that ZNF184

had the closest homology to ZNF204, with 68% nucleic acid ments corresponds to the distance between the phenylalanineresidues of adjoining zinc finger domains of 84 bp. The se-identity over 1963 nucleotides. ZNF184 is also from the

6p21.3 region and within 500 kb of ZNF204 (3, 9). When only quence Lys-Ala-Phe occurs commonly in zinc fingers and canbe encoded by sequences that include a HindIII site. For thethe putative 5* UTR of ZNF204 was analyzed, homology to

the predicted coding ‘‘spacer’’ region of ZNF184 was detected, 10 zinc finger genes with highest homology to ZNF204, 15 ofthe 20 HindIII sites present were positioned at the conservedsuggesting that the 5* region of ZNF204 may have been derived

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phenylalanine residue. Two zinc finger genes contained 5 REFERENCESHindIII sites, all at the phenylalanine residue of the consen-sus, giving rise to a ladder of HindIII fragments as detected 1. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang,by hybridization in Fig. 1d. Conservation of zinc finger probes Z., Miller, W., and Lipman, D. J. (1997). Gapped blast and psi-has been previously reported, with one or several cross-hy- blast: A new generation of protein database search programs.

Nucleic Acids Res. 25: 3389–3402.bridizing bands detected on zoo blots (7). The demonstrationof a conserved ladder of zinc finger fragments by ZNF204 2. Blumberg, H., Eisen, A., Sledziewski, A., Bader, D., and Young,results from the combination of HindIII as a restriction en- E. T. (1987). Two zinc fingers of a yeast regulatory protein

shown by genetic evidence to be essential for its function. Na-zyme, formamide hybridization solution, moderate strin-ture 328: 443–445.gency washing, and possibly the partial degeneracy of the

3. Goldwurm, S., Menzies, M. L., Banyer, J. L., Powell, L. W., andZNF204 probe.Jazwinska, E. C. (1997). Identification of a novel Krueppel-re-We conclude that ZNF204 most likely arose by a local retro-lated zinc finger gene (ZNF184) mapping to 6p21.3. Genomicstransposition event within the 6p21.3 ZNF184 cluster. We40: 486–489.observe a remarkable regular ZNF204 hybridization pattern

4. Pappas, G. J., Polymeropoulos, M. H., Boyle, J. M., and Trent,conserved across all species tested, which we have explainedJ. M. (1995). Regional assignment by hybrid mapping of 36 ex-in terms of the periodicy of HindIII sites within the consensuspressed sequence tags (ESTs) on human chromosome 6. Geno-sequence of multiple zinc finger proteins. At least 35 individ-mics 25: 124–129.ual bands could be resolved in human and bovine DNA, sug-

5. Raha-Chowdhury, R., Bowen, D. J., Stone, C., Pointon, J. J.,gesting that the zinc finger genes, and therefore proteins, ofTerwilliger, J. D., Shearman, J. D., Robson, K. J., Bomford, A.,these species can contain at least 35 motifs. Indeed, the Xfinand Worwood, M. (1995). New polymorphic microsatellite mark-protein of Xenopus contains 37 C2H2 zinc finger motifs, con- ers place the haemochromatosis gene telomeric to D6S105.

sistent with these observations (6). Most zinc finger proteins Hum. Molec. Genet. 4: 1869–1874.are transcriptional regulators involved in cell growth and

6. Ruiz-i-Altaba, A., Perry-O ’Keefe, H., and Melton, D. A. (1987)differentiation. The cross-hybridization of ZNF204 to multi- Xfin: An embryonic gene encoding a multifingered protein inple zinc finger loci across species may provide a tool for the Xenopus. EMBO J. 6: 3065–3070.analysis of C2H2 zinc fingers throughout evolution. 7. Shannon, M., Ashworth, L. K., Mucenski, M. L., Lamerdin,

J. E., Branscomb, E., and Stubbs, L. (1996). Comparative analy-sis of a conserved zinc finger gene cluster on human chromo-ACKNOWLEDGMENTSsome 19q and mouse chromosome 7. Genomics 33: 112–120.

8. Volz, A., Albig, W., Doenecke, D., and Ziegler, A. (1997). Physi-We thank David Latchman, Arian Smit, Dariusz Gorecki, Maritacal mapping of the region around the large histone gene clusterPohlschimidt, Priyal de Zoysa, and Kevin Moore for helpful discus-on human chromosome 6p22.2. DNA Sequence 8: 173–180.sion. The UK HGMP Resource Centre provided PAC clones and the

monochromosomal hybrid panel; the MRC HGMP funded the CCD 9. Wallace, D. F., Partridge, J., Robertson, A., Simpson, V. M. A.,Worwood, M., Bomford, A. B., Volz, A., Ziegler, A., Dooley, J. S.,FISH equipment. This work was funded by grants from The Peter

Samuel Charitable Trust (J.S.D., A.P.W.), and the Nuffield Founda- and Walker, A. P. A 6p22 reference map of leukocyte DNA:Exclusion of rearrangement in four cases of atypical haemochro-tion (A.P.W.). The support of the MRC for the Ph.D. studentship of

J.P. is acknowledged with gratitude. matosis. Eur. J. Hum. Genet., in press.

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