SHORT COMMUNICATION Assignment of the Human 2’,3’-Cyclic Nucleotide 3’-Phosphohydrolase Gene to Chromosome 17 T. J. Spfww.E,*~t Departments

of *Biochemistry

and Molecular Received

K. D. LANCLOS,* AND D. F. LAPP”

Biology and tNeurology, July 17, 1991;

revised

2’,3’-Cyclic nucleotide 3’-phosphohydrolase (CNP) has been used as a general oligodendrocyte and Schwann cell marker enzyme within the nervous system and has been the intense target of a number of recent studies. In this report, we determined the chromosomal localization of the human CNP gene using PCR on two somatic cell DNA panels. PCR amplification, using four primer pairs across an intron, confirms that the CNP gene is localized to chromosome 17. We also present the complete intron sequence of the human gene used to make the assignment. This intron contains a c -+ t polymorphism located at nucleotide 1215, which may be of use in mapping the CNPase gene more precisely within chromosome 17. o ism Academic press, I~C.

Brain contains three major cell types, neurons, astrocytes, and oligodendrocytes. Oligodendrocytes effect the elaboration of myelin membranes that envelope many nerve cell processes. In the peripheral nervous system, myelin synthesis is carried out by the Schwann cell. 2’,3’-Cyclic nucleotide 3’-phosphohydrolase (EC 3.1.4.37, CNPase, CNP) has been shown to be an excellent marker enzyme for both oligodendrocytes and Schwann cells in immunocytochemical studies. The amounts of this myelin-associated enzyme decrease in some neurological mutant animals, under various experimentally induced perturbations of myelin and myelinogenesis, and in a number of neurological disorders in which myelin is affected (review, Ref. (1)). CNP is also located in outer rod segments within the visual system and may be involved in important aspects of membrane biogenesis, elaboration, and function. This enigmatic enzyme hydrolyzes 2’,3’-cyclic nucleotides (and oligonucleotides with a 2’,3’-cyclic terminus) to their corresponding 2’-monophosphates at rates up to over 4000 pmol/min/mg protein. Moreover, the observed in. vitro hydrolysis does not seem to require exogenous Mg2+, Mn2+, or other divalent metal cations. The in vivo function of this enzyme has not been described. The gene was reported to be localized on mouse chromosomes 3 and 11 (2), using coding sequence probes. Because the gene was reported on two chromosomes in mouse, we elected to

Medical February

College of Georgia, Augusta,

Georgia 30912

25, 1992

sequence the human intron 3 and then use intron 3 as a probe to determine the chromosomal assignment in human-hamster somatic hybrids. Two somatic cell (hamster-human) hybrid DNA panels representing a total of 25 different clones were obtained from BIOS Corp. Human chromosomes l-22, X, and Y were represented at least once in each panel. Radioactive nucleotides were obtained from New England Nuclear. DNA-grade agarose and electrophoresis supplies were obtained from Bio-Rad. Tczqpolymerase was purchased from Beckman Instruments. Sequenase Kits were purchased from U.S. Biochemical. Sequencing primers were synthesized as needed on an ABI 340B or an ABI 392. Primer pairs were selected using a modified OLIGO 4.0 program in which relaxation parameters were ranked in their order of application. For amplification and sequencing of the most 3’ human intron, a pair of oligonucleotide primers were designed from the published cDNA sequence (3). The location of the primers were 673 and 1008 bp from the initiation codon, respectively. PCR reactions of genomic DNA yielded a 1.8-kb band that contained the 1.4-kb intervening sequence. Single-stranded DNA used in sequencing was obtained by asymmetric amplification of this region. Nucleotide sequencing was performed by the dideoxy chain-termination method of Sanger (4). Symmetric amplification of the intron 3 region provided double-stranded DNA used in subcloning. Purified intron 3 PCR product was digested with PstI and inserted into PstI-linearized, CIAP-treated pBluescript vector. pBSCNPI3 was purified by CsCl gradient centrifugation. CNP intron 3 insert was released by digestion with PstI, electrophoresed, and then electroeluted from 0.8% agarose gels. The purified fragment was labeled using the Prime-a-gene system of Promega with [(u-32P]dCTP. For the chromosomal assignment of the human CNP gene by PCR, human genomic, CHO genomic, and somatic cell hybrid DNAs were amplified using primers located within the intron, shown underlined in Fig. 1. Reaction mixtures (100 ,ul) containing 100 ng of genomic DNA and 10 pmol of each primer, 20 mM Mops, pH 7.6, 200 PM of each dNTP, 50 mM NaCl, 2.5 mM MgCl, were amplified using 1 unit of Tuq polymerase. Denaturing,

GENOMICS

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Copyright 0 1992 rights of reproduction

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1141 gaggtcagccagcccgctgaggccggaagccagtgtgcagggagctgggctccctcagga 1201 agtccggctgtcccrgtgtggccctgtctgagcgccttcgtgtttctcacttgagcctgg 1261 agcgcctatggaaggtgcgtgccgcagttgttagtggatgctgggcctataccgacccct 1321 gctcagctgtgctcacttctgttagacttccccttctctcctccaggagggaccctccct 1381 gggtcagctgttcctcaagagcctgccatctctagcttgggcccctctttctcactctcc 1441 ccag FIG. 1. Completemostdownstream intronsequenceofthehuman 2',3'-cyclicnucleotide3'-phosphohydrolasegene.HumangenomicDNA wassequencedacrossexons3and4.The complete intronis 1444bpinlengthandcontainsaG-richregionnearthebeginningofthesequence. PCR amplification across this intron sequence was then used to determine the chromosomal localization of the gene in somatic ce11hybrid DNAs.Theinitial primer pairusedto amplifythisintroninthis reportisunderlined. A polymorphic site (C -+~)wasidentifiedatbase1215and is also underlined.

annealing, and extension temperatures and times were 94°C for 1 min, 58°C for 1.5 min, and 72°C for 3 min for the 958-bp PCR product, respectively. Nested primer pairs for 478-, 442-, and lSl-bp products within this intron were also used with a hot start of 95°C for 4 min, addition of hot dNTPs in a volume of 30 ~1 through the mineral oil layer over 1 min, and then a 33- to 35-cycle two-temperature PCR at 94°C for 1 min and 60°C for 1.5 min. With all three sets of primers, 50 ng of total DNA and 20 pmol of each primer were used in the incubation mixtures. Ten-microliter aliquots of each reaction mixture were electrophoresed on 0.8-1.0% agarose gels. To determine the appropriate restriction enzyme digest in preliminary experiments, 5 pg of human DNA and 5 pugof hamster DNA controls were each digested with EcoRI, HindIII, and BarnHI, and Southern blots were probed with labeled intron 3 fragment (958 bp). In

subsequent experiments, 5 fig of chromosomal DNA from human-hamster somatic cell Panel I and 5 pg of their normal controls were digested with EcoRI, electrophoresed in 0.8% agarose gels, and transferred to nylon membranes. Membranes were prehybridized in 6X SSC, 5~ Denhardt’s, 0.5% SDS, and 100 pg/ml salmon sperm DNA for 4 h at 65°C. 32P-labeled probe (478-bp product, nested intron primers) was added and incubation continued overnight. Washing progressed from 2~ SSC, 0.5% SDS at 25°C through 0.1X SSC, 0.5% SDS at 60°C for 30 min and membranes were exposed to X-ray film to further confirm the PCR assignments. The complete intron 3 sequence of human 2’,3’-cyclic nucleotide 3’-phosphohydrolase is shown in Fig. 1. This sequence is fully contiguous with the coding sequences of exons 3 and 4 (not shown) (3). Both the intron 5’ (GT) and the 3’ sequences (AG) are characteristic of eukary-

SHORT

gene. Somatic cell DNA from panel I, digested with EcoRI, blotted, and probed with 32P-labeled intron, resulted in labeling of the hybrid containing chromosome 17 (SM 811, not shown). The results of preliminary Southern blots of Panel I are consistent with the PCR assignment of chromosome 17 in both somatic cell hybrid DNA panels. The specific somatic cell hybrids analyzed in this report are summarized in Table 1. Four distinct nested primer pairs within this same CNP intron amplified the expected 958-, 478-, 442-, and 181-bp products within this intron in both somatic cell DNA panels. In Fig. 2, for example, an agarose gel of a twotemperature PCR (50 ng of each DNA, 478-bp product) with the unique hybrids of panels I and II (BIOS) is shown. The numbers correspond to the gel lane numbers. Ladder DNA size markers were applied to lanes 1 and 30. Lanes 11 and 18 contain PCR product from two distinct hybrids, each of which contain human chromosome 17. Lanes 27 and 29 contained control human genomic DNA, and lane 28 contained host hamster DNA, which did not amplify. In humans, CNP has been reported to occur in two

otic intron-exon junctions. In the human gene, this is the most 3’ intron. The primer pair selected for amplification (958-bp product) is underlined. The site of the observed c + t polymorphism is also underlined at nucleotide 1215. PCR amplification of both somatic cell hybrid DNA panels produced the expected product only in clones containing chromosome 17. SM 811 hybrid in panel I contained human chromosomes 8, 17, and 18. However, chromosome 8 was also represented in lanes 6, 11, 13, and 16, none of which amplified. Similarly, chromosome 18 was also contained in lanes 1, 4, and 11, which also did not amplify. In PCR panel II, one lane on the agarose gel contained (SM 937) human chromosomes 1,5,14,15,17, and 21. These chromosomes, other than 17, were represented in 2-16 samples in the 18member DNA panel; none of these other human chromosomes amplified with our primers. Host (hamster) chromosomal DNA did not amplify at all, suggesting extensive mismatches with one or both primers. In each panel, human genomic DNA produced the expected intron product. We, therefore, conclude from the PCR data that chromosome 17 contains the human CNP

TABLE Summary

of PCR Amplification

of the Two

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FIG. 2. Two-temperature PCR across a polymorphic site on the human CNP gene with two somatic cell DNA panels. Nested primers across the polymorphic site at nucleotide 1215 (see Fig. 1) were used to determine the chromosome(s) on which the CNP gene was located. PCR was carried out at two temperatures (94 and 60°C) using a modified hot start, 50 ng of DNA, and 20 pmol of primer as described above. Ten-microliter aliquots of the reaction mixture were applied to a 1% agarose gel and electrophoresed in 1~ TBE buffer at 45 V. The gel was then stained with ethidium bromide to identify the 478-bp product. Ladder DNA size markers are shown in lanes 1 and 30. Human control genomic DNA reaction mixtures were applied to lanes 27 and 29. Hamster control reaction mixture was applied to lane 28. Lanes 11 and 18 correspond to hybrids SM 811 and SM 937, respectively, as referred to in Table 1.

isoforms approximately 46 and 48 kDa in size (1). The gions of the gene, and its expression in cell culture and in difference between the two CNP forms could arise from transgenic mice. alternative splicing of the very basic, serine-rich 19-20 ACKNOWLEDGMENTS amino acids on the amino terminus of the protein. Another plausible explanation is the existence of a second This work was supported by Grants BNS-8916771 (NSF), NSstart site in the gene. The amino acid sequence of this 18279 (NIH), and RG1718-B-2 (NMSS) to T.J.S., and by the Vetlarger, more basic 48-kDa form may explain the differerans Administration. The authors acknowledge the technical support ential phosphorylation of this enzyme observed at least of Sarah Wall and Janette Roberts for typing the manuscript. Sequence data from this article have been deposited with the EMBL/ in rodents, where the 4%kDa protein is phosphorylated Data Libraries under Accession No. X56614 for human in uiuo about lo-fold higher than the 46-kDa protein on a GenBank HIJCNPASE DNA. mass basis (5). Speculation about the physiological function and properties of this myelin-associated enREFERENCES zyme may include a role in signal transduction, its associ1. Sprinkle, T. d. (1989). “CRC Critical Reviews in Neurobiology,” ation with cytoskeletal elements, and a role in nucleic Vol. 4, pp. 2355301, CRC Press, Boca Raton, FL. acid metabolism, possibly RNA. Further studies are 2. Bernier. L., Colman, D. R., and DEustachio, P. (1988). J. Neuroneeded to determine the gene structure, its location sci. Res. 20, 497-504. within chromosome 17, and its expression. These stud3. Kurihara, T., Takahashi, Y., Nishiyama, A., and Kumanishi, T. ies should aid in elucidating the physiological function of (1988). Biochem. Biophys. Res. Commun. 152,837-891. this enzyme. Some of our current investigations focus on 4. Sanger, F., Nicklen, S., and Coulson, A. R. (1977). Proc. Natl. the splicing patterns, gene copy number, regulatory seAcad. Sci. USA 74,5463-5467. quences, breakpoint panel chromosomal mapping, site5. Agrawal, H. C., Sprinkle, T. J., and Agrawal, D. (1990). Biochem. Biophys. Res. Commun. 170,817-823. directed alterations to specific nucleotide binding re-