Proc. Nadl. Acad. Sci. USA Vol. 89, pp. 5457-5461, June 1992 Medical Sciences A putative gene family in 15q11-13 and 16pll.2: Possible implications for Prader-Willi and Angelman syndromes KARIN BuITING*, VALERIE GREGER*, BERNHARD H. BROWNSTEINt, ROSE M. MOHRt, ION VOICULESCuO, ANDREAS WINTERPACHT§, BERNHARD ZABEL§, AND BERNHARD HORSTHEMKE*¶ *Institut for Humangenetik, Universititsklinikum Essen, 4300 Essen 1, Federal Republic of Germany; tCenter for Genetics in Medicine, Washington University, St. Louis, MO 63110; tInstitut fOr Humangenetik und Anthropologie, Universitit Freiburg, 7800 Freiburg, Federal Republic of Germany; and §Kinderklinik, UniversitAt Mainz, 6500 Mainz, Federal Republic of Germany Communicated by Victor A. McKusick, February 13, 1992

ABSTRACT The genetic defects in Prader-Willi syndrome of mouse A9 cells with lymphoblastoid cells from PWS (PWS) and Angelman syndrome (AS) map to 15q11-13. Using patient 168 (14) as described by Wigler et al. (15). The somatic microdissection, we have recently isolated several DNA probes cell hybrid mapping panels were kindly provided by G. Bruns for the critical region. Here we report that microclone MN7 (Boston) (panel I) and K. H. Grzeschik (Marburg) (panel II). detects multiple loci in 15q11-13 and 16pll.2. Eight yeast Genomic DNA Analysis. Genomic DNA was analyzed by artificial chromosome (YAC) clones, two genomic phage Southern blot hybridization as described (16). The final wash clones, and two placenta cDNA dones were isolated to analyze was usually at 650C in 150 mM sodium chloride/15 mM these loci in detail. Two ofthe YAC clones map to 16p. Six YAC sodium citrate (lx SSC) containing 0.1% SDS. The following clones and two genomic phage clones contain a total of four or probes were used: HuD2 (IGHDY2) (17), MN60 (D15564) five different MN7 copies, which are spread over a large (14), MN8 (D15S77) (14), MN47 (D15S78) (14), MN43 distance within 15q11-13. One cDNA clone is from chromo- (D15S80) (14), MN28 (D15S81) (14), PW66 (D15S79) (14), some 15 and one is from chromosome 16. The chromosome 15 PW71 (D15S63) (14), ML34 (D15S9) (13), 3-21 (D15S10) (13), cDNA detects transcripts of 14 and 8 kilobases in various IR4-3R (D15S11) (13), IR10-1 (D15S12) (13), IR39 (D15S18) human tissues. The presence ofmultiple copies ofthe MN7 gene (13), 28,33-H3 (GABRB3) (18), CMW1 (D15S24) (19), c-fes family in proximal 15q may conceivably be related to the (fps/fes protooncogene). ML34, 3-21, IR4-3R, IR10-1, IR39, instability of this region and thus to the etiology of assoiated and 28p3-H3 were kindly provided by M. Lalande (Boston). disorders. HuD2 was a gift from P. Leder (Boston). CMW1 was kindly provided by D. Ledbetter (Houston). c-fes was from Amer- sham. For PCR analysis of genomic Bgl II fragments, the The Prader-Willi syndrome (PWS) is a complex disorder fragments were separated on low-melting-point agarose gels, whose major features include severe infantile hypotonia, excised from the gel, and purified with Geneclean (Bio 101, failure to thrive, hypothalamic dysfunction causing genital La Jolla, CA). hypoplasia and pubertal insufficiency, hyperphagia with obe- DNA Amplification. PCR primers were synthesized on an sity developing after the neonatal period, small hands and Applied Biosystems DNA synthesizer (model 391). Amplifi- feet, short stature, and mental retardation (1). PWS occurs in cation was carried out with the help of a thermal cycler -1/25,000 live births. About 60%o of the PWS patients have (Perkin-Elmer/Cetus) in a total vol of 100 jul containing 50 deletions or other chromosomal abnormalities (transloca- mM KCl, 10 mM Tris-HCl (pH 8.0), 1.5 mM MgCI2, 0.01% tions or duplications) on the proximal long arm of chromo- gelatin, 200 ,uM each dNTP, and 2.0 units of Taq DNA some 15 (15q11.2-12) (2, 3). Interestingly, similar deletions polymerase. MN7 sequences were amplified with 0.5 ,uM are found in "50% ofpatients with Angelman syndrome (AS) primers MN7a and MN7b (see below) from 10 ng of genomic (4-8). AS is characterized by mental retardation, jerky DNA or 100 ng of total yeast DNA in 35 cycles of denatur- movements, paroxysms of laughter, protruding tongue, a ation (940C for 1 min), annealing (520C for 2 min), and characteristic facial appearance, microcephaly, and an ab- extension (72°C for 3 min). The samples were boiled for 5 min normal electroencephalogram pattern (9). The deletion in before Taq DNA polymerase was added. Interspersed repet- PWS patients is of paternal origin (10), whereas deletions in itive sequence (IRS) PCR (20) was carried out with 1 ,ug of AS are of maternal origin (11). Findings of maternal disomy total yeast DNA for 35 cycles of denaturation (94°C for 1 of chromosome 15q11-13 in PWS patients (11) and paternal min), annealing (55°C for 1 min), and extension (72°C for 4 disomy in AS patients (12) suggest that genomic imprinting min). The samples were boiled for 10 min before Taq DNA affects the expression of genes in proximal 15q. As the polymerase was added. Primer 517 was used at 0.3 ,uM, and biochemical defects in both syndromes are unknown, posi- primer PDJ34 was used at 0.1 ,AM. PCR primer sequences are tional cloning has to be used to identify these genes. Donlon as follows: MN7a, 5'-ACCTGATCGCCCCATTTCCA-3'; et al. (13) used flow sorting to isolate DNA markers for MN7b, 5'-ACTTTGTGAATGTTTTGTGT-3'; 517, 5'- proximal 15q. Using microdissection and microcloning of CGACCTCGAGATCT(C/T)(G/A)GCTCACTGCAA-3'; banded metaphase chromosomes, we have recently isolated PDJ34, 5'-TGAGC(C/T)(G/A)(A/T)GAT(C/T)(G/A)(C/ several clones from this region (14). Here we report the T)(C/A)CCA(C/T)TGCACTCCAGCCTGGG-3'. detailed characterization of the microclone MN7. Isolation of Yeast Artificial Chromosomes (YACs) and Ge- nomic Phage Clones. Screening the YAC library ofthe Center MATERIALS AND METHODS for Genetics in Medicine (Washington University) for YACs containing cognate sequences for MN7 was performed es- Somatic Cell Hybridization. Somatic cell hybrids Hz16 and sentially as described by Brownstein et al. (21) and by Green Hz31 were obtained by polyethylene glycol-stimulated fusion Abbreviations: PWS, Prader-Willi syndrome; AS, Angelman syn- The publication costs of this article were defrayed in part by page charge drome; IRS, interspersed repetitive sequence; YAC, yeast artificial payment. This article must therefore be hereby marked "advertisement" chromosome; TAFE, transverse alternating field electrophoresis. in accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed.

5457 Downloaded by guest on October 4, 2021 5458 Medical Sciences: Buiting et al. Proc. Natl. Acad. Sci. USA 89 (1992) and Olson (22). A human genomic phage library in AL47.1 Table 1. YACs containing cognate sequences for MN7 (23) was screened by hybridization with MN7. Among Fragment 500,000 clones screened, 12 positive plaques were identified. containing Two clones (A7.7 and A7.12) were plaque purified in two rounds. Size, MN7, kb Chromo- Pulsed-Field Gel Analysis. High molecular weight yeast YAC Name kb Bgi II Dra I some DNA in agarose plugs was prepared as described by Carle 1 A86C1 600 9.0 3.0 15 and Olson (24). Yeast chromosomes were separated by 5.0 transverse alternating field electrophoresis (TAFE; Beck- 2 A162B10 310 9.0 3.0 15 man) in 10 mM Tris HCI/0.5 mM EDTA (free acid)/4.4 mM 3 A208G12 260 9.0 3.0 15 acetic acid, pH 8.2. Running conditions were 1 hr with 4-sec 4 A137C11 180 9.0 3.0 15 pulses at 170 mA followed by 21 hr with 50-sec pulses at 150 5 A85A5 180 9.0 3.0 15 mA or 22 hr with 25-sec pulses at 150 mA at 14°C buffer 6 A31D2 350 9.0 3.0 15 temperature. DNA was stained with ethidium bromide. Sac- 7 A85H4 250 9.0 1.3 16 charomyces cerevisiae chromosomes (strain 334; Beckman) 8 A28F11 300 9.0 1.3 16 were used as size markers. Isolation and Characterization of cDNA Clones. Approxi- RESULTS mately 1 x 106 phages from a human placenta 5'-Stretch Genomic DNA Analysis and Somatic Cell Genetics. MN7 is a cDNA library (Clontech) were screened with probe MN7. Six 275-base-pair (bp) microclone, which does not contain recog- positive clones were found, and two clones (c7.541 and nition sites for Dra I and Bgl II. Southern blot analysis ofDra c7.681) were plaque purified. c7.541 was used to probe a I- and Bgl II-digested human DNA with MN7, however, human multiple tissue Northern (MTN) blot (Clontech) con- revealed multiple bands (Fig. 1, lanes ME). In Dra I-digested taining 2 ,ug of poly(A)+ RNA from placenta and adult brain, DNA, two fragments of 3.0 and 1.3 kilobases (kb) are ob- heart, liver, kidney, pancreas, and skeletal muscle. Final served. The numberofBgl II fragments varies between normal washing of the filter was at 50°C in O.lx SSC/0.1% SDS. individuals. Of 12 unrelated individuals tested, 12 had the 9.0-, In Situ Chromosome Hybridization. The cDNA c7.681 was 5.0-, and 3.0-kb Bgl II fragment; one had the 20-kb fragment; 3H-labeled by the random priming method (5 x 107 cpm per and nine had the 5.5-kb fragment (data not shown). For ,ug of DNA) and hybridized to normal human lymphocyte chromosomal localization of these fragments, we screened (pro)metaphase chromosomes as described (25). two somatic cell hybrid mapping panels. The results obtained Sequence Analysis. PCR product (20 ng) was purified on with panel I are shown in Fig. 1. The 3.0-kb Dra I band maps low-melting-point agarose gels and Ultrafree-MC filter tubes to chromosome 15 and the 1.3-kb Dra I band maps to chro- (Millipore). DNA was sequenced with 50 pmol of primers mosome 16. The 9.0-kb Bgl II band contains DNA from both MN7a and MN7b. Pst I fragments of the cDNA clones were chromosomes 15 and 16. The 5.0- and 3.0-kb Bgl II bands map subcloned into vectors pT7T3 or pUC19. Amplification prod- to chromosome 15. Because of their polymorphic nature, the ucts and-cDNA subclones were sequenced by the dideoxy- 20- and 5.5-kb bands cannot be assigned unambiguously. The nucleotide chain-termination method and Sequenase (United results, however, suggest that the 20-kb band maps to chro- States Biochemical). mosome 16 and the 5.5-kb band maps to chromosome 15. Chromosome *

1 5 _

16 +-- -

- Zt CO Ln LL < U- Q LL < LL 2 LO) L) U LO CM U) Lr C CO C:) CO CLCU 2 kb CD) r r1, 71 _ __0 _p _ - 20.0 -9.0 W UVIP

5 .=I 0, a~~~--5.

3.0 11 PW - hamster qw _ 1 Cr '

BgL Dra.

FIG. 1. Chromosomal assignment of MN7 sequences. Southern blots containing BgI II- and Dra I-digested DNA from hamster-human cell hybrids (G35F1, G35A4, G35F3, G35D5, CP22, and 15A) and a normal human control DNA (ME) were hybridized with 32P-labeled MN7. Downloaded by guest on October 4, 2021 Medical Sciences: Buiting et al. Proc. Natl. Acad. Sci. USA 89 (1992) 5459 YAC 1 CMW1, MN60, MN28, GABRB3, and c-fes. This indicates that Hz16 contains a fragment of the normal chromosome 15. o o Southern blot analysis of these two cell lines with MN7 kb MS°n revealed that Hz31 contains the 20- and the 9.0-kb Bgl II fragment, indicating that most of the chromosome 15 MN7 1.6 - loci are deleted in patient 168. Hz16 contains the 3.0- and the 5.5-kb Bgi II fragment. This suggests that the distal break- 1.0 -- point of the chromosome 15 fragment in Hz16 lies within the FIG. 2. IRS PCR analysis of critical PWS region and the MN7 cluster. 0.5 YAC 1. The 600- and the 350-kb Isolation of Genomic Clones. To clone the genomic envi- YACs of A86C1 (YAC 1) were ronment of MN7, we screened a genomic phage library and 0.3 - separated by TAFE and analyzed a YAC library. Two phage clones of 15 kb (A7.7 and A7.12) with primers PDJ34 and 517. M, and eight YACs (Table 1) were obtained. In the two phage marker. clones, MN7 is contained on a 3.0-kb Dra I fragment. This indicates that the clones are derived from chromosome 15 Regional Mapping ofMN7 Gene Copies. To map the different (see above). MN7 loci with respect to the critical PWS region, we have isolated the deletion chromosome 15 from PWS patient 168 (14) in mouse-human cell hybrids. Southern blot analysis ; *#'' showed that one cell line (Hz3l) is positive for HuD2, IR39, \0 _. A ML34, MN60, MN28, and c-fes and negative for 3-21, 4-3R, IR10, PW71, PW66, MN8, MN47, MN43, CMW1, and GA- la BRB3. This confirms previous results obtained by quantitative Southern blot analysis of patient DNA and indicates that cell I"I'*8 *~\15 ~ ..f%% line Hz31 contains deletion chromosome 15. Hybrid cell line 11*# Hz16 was positive for markers HuD2, IR39, ML34, 3-21, 4-3R, b and PW71 and negative for IR10, PW66, MN8, MN47, MN43, as A _r t c: U) c 0M/Mm. _ 7.681

wh o 0 u L CL CL oL u u Lll LU LL

- -Fj E=! n Ec: c7.541

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.. B .0 CD :::m ~~m~E U) _ -0- >% a) a 0 CZ ia C) w er. -- Q0 -a - > r- kb

- 14 I0e.....

-. J..:.:.:' _~~ ^:V. 16 FIG. 4. Chromosome in situ hybridization. (Upper) Partial FIG. 3. cDNA and mRNA analysis. (A) Restriction map of MN7 G-banded metaphase showing both chromosomes 15 labeled at cDNA clones c7.681 and c7.541. Hatched boxes indicate regions of 15q11-12 and silver grains on chromosomes 16 at 16pll.2. (Lower) homology to MN7. (B) Northern blot analysis. Poly(A)+ RNA from Idiogram ofchromosomes 15 and 16 showing silver grain distribution several tissues was hybridized with the 32P-labeled cDNA clone on chromosomes 15 and 16 from 30 metaphases with specific labeling c7.541. of 15q11 and 16pll.2. Downloaded by guest on October 4, 2021 5460 Medical Sciences: Buiting et al. Proc. Natl. Acad. Sci. USA 89 (1992)

C T c >- G C CG AC C TA G T A MN7 microclone G y 2, 4, 6, 1(9kb-Bgl II);X7.12; Hz 16 genomic DNA (5.5kb-Bgl 11) A G y 1(5kb-Bgl 11) G C G y3,5; Hz 31 genomic DNA (9kb-Bgl 11) G G genomic DNA (3kb-Bgl 11); A 7.7 G G cDNA 7.541 T T T T GC T C G A T y7 A T T T T GC T C G y8 T GC T C G cDNA 7.681 FIG. 5. Comparison of the MN7 sequence in genomic DNA, phage clones, YAC clones, and homologous region of the cDNA clones. For clarity, only nucleotide differences are indicated. For mapping the YACs, total yeast DNA was digested with (Fig. 4). No other chromosome demonstrated grain counts Bgl II and Dra I and probed with MN7. MN7 detects a 9.0- and above background level. a 5.0-kb Bgl II fragment and a 3.0-kb Dra I fragment in YAC Sequence Analysis. Except for a few different bases, 188 bp 1, a 9.0-kb Bgl II fragment and a 3.0-kb Dra I fragment in of both cDNAs are identical to the microclone MN7. To YACs 2-6, and a 9.0-kb Bgl II fragment and a 1.3-kb Dra I determine from which ofthe various MN7 loci the cDNAs are fragment in YACs 7 and 8 (Table 1). According to somatic cell derived, we amplified the MN7 sequence in the phage and genetic analysis (Fig. 1), YACs 1-6 originate from chromo- YAC clones as well as in the 5.5-kb Bgl II fragment from the some 15 and YACs 7 and 8 originate from chromosome 16. hybrid cell line Hz16, the 9.0-kb Bgl II fragment from hybrid In subsequent preparations of YAC 1, it was noticed that cell line Hz31, and the genomic 3.0-kb Bgl II fragment from the 600-kb hybridization signal was very weak and a 350-kb normal control DNA. All sequences differ from the micro- YAC that did not hybridize to MN7 was present. To inves- clone by a T to G exchange (Fig. 5). It is possible that this represents an error of the DNA polymerase used for PCR- tigate whether these two YACs were related or were cotrans- mediated microcloning. The homologous region of c7.541 is formation products, we isolated both YACs by preparative identical to phage A7.7 and the 3.0-kb genomic fragment, TAFE. Inter-Alu PCR with primers PDJ34 and 517 gave two which map to chromosome 15. The homologous region in PCR products of identical size (Fig. 2). This suggests that the c7.681 is identical to YAC 8, which maps to chromosome 16. 350-kb YAC may be derived from the 600-kb YAC. Northern Blot Analysis. To study the tissue distribution of Isolation and Analysis of cDNA Clones. As reported previ- MN7 transcripts, poly(A)+ RNA from placenta and from ously, MN7 is evolutionarily conserved (14). To investigate adult brain, heart, lung, liver, kidney, pancreas, and skeletal whether MN7 contains gene sequences, we screened a hu- muscle was analyzed by Northern blot hybridization with man placental cDNA library. Two partial cDNA clones of 1.6 c7.541. In most tissues, transcripts of 8 and 14 kb were (c7.681) and 2.0 (c7.541) kb were obtained. Restriction map- detected (Fig. 3B). In some tissues, additional signals ofweak ping revealed considerable differences between the two intensity were observed. clones (Fig. 3A). The chromosomal assignment ofc7.681 was done by the technique of in situ hybridization. Analysis of DISCUSSION silver grain distribution in 30 metaphases localized 24% of Microclone MN7 detects multiple restriction fragments, total grains on chromosome 15 and 23% on chromosome 16. which we have mapped to 15q11-13 and 16p11.2 by somatic More than 80% of the grains found on these chromosomes cell genetics and chromosome in situ hybridization. Apart identified a specific area (15qll-12 and 16pll.2, respectively) from the 20- and 5.5-kb Bgl II fragments, all fragments were Locus (Probe) Hybrid cell line MN7 BglII Clone MN7 Locus Hz31 Hzl6 fragment (kb) IGDHY2 (HuD2) Dl5S9 (ML34) Dl5S18 (IR39) DI S 10 (3-21) 3.0 X7.7 D15F37S4E D15S~l (4-3R) _ 9.0/5.5 y2,4,6; X7.12 DI5F37S2 FIG. 6. Tentative localization DI 5S36 (PW71)x of MN7 loci within proximal 15q. GABRB3 (28B3-H3) 9.0 yl DI5F37SI DNA from cell hybrids Hz16 and D15S12 (IR1O) 5.0 yl D15F37S1 Hz31 was probed with MN7 and D15S24 (CMWI) the markers listed on the left. D15S77 (MN8) Solid bar indicates the presence of D15S78 (MN47) a marker. As we cannot rule out Dl5S79 (PW66) rearrangements of the chromo- D15S80 (MN43) some 15 DNA in the patient or in D15S64 (MN60) 9.0 y3,5 D15F37S3 the cell hybrids, the probe order D15S81 (MN28) should be regarded as tentative. FES (c-fes) Within each map interval, the probes are listed arbitrarily. Downloaded by guest on October 4, 2021 Medical Sciences: Buiting et al. Proc. Natl. Acad. Sci. USA 89 (1992) 5461 isolated in phage or YAC clones. Restriction enzyme analysis 1. Prader, A., Labhart, A. & Willi, H. (1956) Schweiz. Med. and partial sequencing indicate that YACs 7 and 8 are from Wochenschr. 86, 1260-1261. chromosome 16. YACs 1-6 and A7.7 and A7.12 are from 2. Ledbetter, D. H., Greenberg, F., Holm, V. A. & Cassidy, chromosome 15. S. B. (1987) Am. J. Med. Genet. 28, 779-790. YACs 2, 4, and 6 and YACs 3 and 5 form two contiguous 3. Butler, M. G. (1990) Am. J. Med. Genet. 35, 319-332. 4. Kaplan, L. C., Wharton, R., Elias, E., Mandell, F., Donlon, sequences, which contain two different MN7 sequences. The T. A. & Latt, S. A. (1987) Am. J. Med. Genet. 28, 45-53. MN7 sequence in the 9.0-kb Bgl II fragment of YAC 1 is 5. Magenis, R. E., Brown, M. G., Lacy, D. A., Budden, S. & identical to the MN7 sequence in YACs 2,4, and 6. However, Fafranchi, S. (1987) Am. J. Med. Genet. 28, 829-838. YAC 1 does not share any IRS PCR product with YACs 2, 4, 6. Pembrey, M., Fennell, S. J., Van Den Berghe, J., Fitchett, M., and 6 (data not shown). Furthermore, the 5.0-kb Bgl II Summers, D., Butler, L., Clarke, C., Griffith, M., Thompson, fragment on YAC 1 is absent in the Hz16 cell hybrid. This E., Super, M. & Baraitser, M. (1989) J. Med. Genet. 26, 73-77. suggests that unless YAC 1 spans the breakpoint in Hz16, the 7. Knoll, J. H. M., Nicholls, R. D., Magenis, R. E., Graham, two 9.0-kb Bgl II fragments are derived from two different J. M., Jr., Lalande, M. & Latt, S. A. (1989) Am. J. Med. Genet. loci. Sequence analysis ofthe 5.5-kb Bgl II fragment in Hz16 32, 285-290. suggests that this fragment is allelic to a 9.0-kb Bgl II 8. Williams, C. A., Gray, B. A., Hendrickson, J. E., Stone, J. W. fragment. Thus, there are four or five MN7 copies spread & Cantu, E. S. (1989) Am. J. Med. Genet. 32, 339-345. over a large distance within 15qll-13 (Fig. 6). Apart from the 9. Angelman, H. (1965) Dev. Med. Neurol. 7, 681-688. 10. Butler, M. G., Meaney, F. J. & Palmer, C. G. (1986) Am. J. most distal copy, all chromosome 15 MN7 copies are deleted Med. Genet. 23, 793-809. in PWS patient 168, who has acytogenetically visible deletion 11. Nicholls, R. D., Knoll, J. H. M., Butler, M. G., Karam, S. & of chromosome 15. Lalande, M. (1989) Nature (London) 342, 281-285. YAC 1, which contains two MN7 loci on a 600-kb insert, 12. Malcom, S., Clayton-Smith, J., Nichols, M., Robb, S., Webb, appears to be unstable in yeast cells. It is possible that the T., Armour, J. A. L., Jeffreys, A. J. & Pembrey, M. E. (1991) instability of YAC 1 is caused by the presence of two MN7 Lancet 337, 694-697. repeat units and that the presence of multiple copies of this 13. Donlon, T. A., Lalande, M., Wyman, A., Bruns, G. & Latt, sequence in proximal 15q may be related to the instability of S. A. (1986) Proc. Natl. Acad. Sci. USA 83, 4408-4412, and this region and associated clinical disorders. correction (1986) 83, 6964. The chromosome 15-specific MN7 sequences differ by 14. Buiting, K., Neumann, M., Ludecke, H. J., Senger, G., Claus- only 1% of nucleotide exchanges. This is similar to the sen, U., Antich, J., Passarge, E. & Horsthemke, B. (1990) MN7 Genomics 6, 521-527. differences observed in the chromosome 16-specific 15. Wigler, M., Pellicer, A., Silverstein, S., Axel, R., Urlaub, G. sequences (Fig. 5). However, the chromosome 15 sequences & Chasin, L. (1979) Proc. Nati. Acad. Sci. USA 76, 1373-1376. differ from those of chromosome 16 by 5% of nucleotide 16. Horsthemke, B., Greger, V., Barnert, H. J., Hopping, W. & exchanges. In mouse, MN7 detects a single locus on mouse Passarge, E. (1987) Hum. Genet. 76, 257-261. chromosome 7, which contains a region of homology with 17. Siebenlist, U., Ravetch, J. V., Korsmeyer, S., Waldmann, T. human chromosome 15 (26, 27). This suggests that the & Leder, P. (1981) Nature (London) 294, 631-635. multiple MN7 loci in humans are the product of gene dupli- 18. Wagstaff, J., Knoll, J. H. M., Fleming, J., Kirkness, E. F., cation events that occurred after the evolutionary divergence Martin-Gallardo, A., Greenberg, F., Graham, J. M., Jr., Men- of rodents and primates. As the interchromosomal sequence ninger, J., Ward, D., Venter, C. & Lalande, M. (1991) Am. J. than the intrachromosomal sequence Hum. Genet. 49, 330-337. difference is larger 19. Rich, D. C., Witkowski, C. M., Summers, K. M., van Tuinen, difference, the transposition of the MN7 sequence to chro- P. & Ledbetter, D. H. (1988) Nucleic Acids Res. 16, 8740. mosome 16 occurred at an early stage of MN7 duplication. 20. Nelson, D. L., Ledbetter, S. A., Corbo, L., Victoria, M. F., The large interchromosomal difference of the MN7 se- Ramirez-Solis, R., Webster, T. D., Ledbetter, D. H. & Cas- quence has made it possible to assign cDNA 7.541 to chro- key, C. T. (1989) Proc. Nati. Acad. Sci. USA 86, 6686-6690. mosome 15 and cDNA 7.681 to chromosome 16. Complete 21. Brownstein, B. H., Silverman, G. A., Little, R. D., Burke, sequence identity ofthe homologous MN7 sequence in c7.541 D. T., Korsmeyer, S. J., Schlessinger, D. & Olson, M. V. with the 3.0-kb genomic Bgl II fragment suggests that the (1989) Science 244, 1348-1351. mRNA was transcribed from this locus. In Northern blot 22. Green, D. E. & Olson, M. V. (1991) Proc. Nati. Acad. Sci. c7.541 detects at least two transcripts in several USA 87, 1213-1217. analysis, 23. Ludecke, H. J. L., Johnson, C., Wagner, M., Wells, D., Tur- tissues, indicating expression of several loci or alternative leau, C., Tommerup, N., Latos-Bielenska, A., Sandig, K. R., splicing. Since the cDNA sequences have no apparent ho- Meinecke, P., Zabel, B. & Horsthemke, B. (1991) Am. J. Hum. mology to other known human genes, the function of the Genet. 49, 1197-1206. MN7 gene family remains unknown. 24. Carle, G. F. & Olson, M. V. (1984) Nucleic Acids Res. 12, 5647-5664. We thank I. Horstmann, B. Brandt, and S. Gross for expert 25. Naylor, S. L., McGill, R. & Zabel, B. U. (1987) Methods technical assistance; Dr. M. Lalande and Dr. G. Scherer for helpful Enzymol. 151, 279-292. discussions; J. Zimmer and Dr. J. H. M. Knoll for somatic cell 26. Chaillet, J. R., Knoll, H. J. M., Horsthemke, B. & Lalande, hybrid analysis; Dr. D. Schlessinger for critical reading of the M. (1991) Genomics 11, 773-776. manuscript; and Prof. E. Passarge for continuous support. Parts of 27. Nicholls, R. D., Horsthemke, B. & Neuman, P. (1991) Mouse this work were supported by the Deutsche Forschungsgemeinschaft. Genome 89, 254. Downloaded by guest on October 4, 2021