Mapping of a newly discovered human gene homologous to the apoptosis associated-murine mammary protein, MFG-E8, to chromosome 15q25.

conserved matches with the mouse protein. The sequence Mapping of a Newly Discovered extends 20 bases into a 3* untranslated region. We call this cDNA huMFG-E8. Larocca et al. (4) have reported a partial Human Gene Homologous to the cDNA sequence, obtained from a human breast tumor, that Apoptosis Associated-Murine identically matches with hMFG-E8 from nucleotide 514 (huMFG-E8 numbering) to 1190 and then extends an addiMammary Protein, MFG-E8, tional 600 bases into a 3* untranslated region. Larocca et al. to Chromosome 15q25 (4) have also observed a mRNA corresponding to this cDNA to be significantly higher than in normal breast tissue in Colin Collins,* Jan O. Nehlin,* John D. Stubbs,† Northern blots of two human breast tissue carcinoma lines David Kowbel,* Wen-Lin Kuo,‡ and G. Parry§ and a human ovary carcinoma cell line. A single PAC clone was isolated by screening a human *Berkeley National Laboratory, Berkeley, California 94720; †Department of Biological Sciences, San Francisco State University, San Francisco, PAC library arrayed on high-density filters (Genome SysCalifornia 94132; ‡University of California, San Francisco, tems). Prehybridization and hybridization were performed San Francisco, California 94117; and §Berlex Biosciences, according to the Genome Systems protocol. A 425-bp hybridRichmond, California 94804 ization probe was generated by amplification from huMFGE8 using the PCR in glass capillary tubes (Idaho TechnoloReceived July 22, 1996; accepted September 30, 1996 gies). PCR was performed in 10-ml volumes containing 10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 250 mg/ ml BSA (Gibco-BRL), 100 mM for each dNTP, 10 pmol of each primer, and 0.5U AmpliTaq. An amplification product from We report here the identification and mapping of a human the huMFG-E8 cDNA template was observed using 35 PCR PAC clone containing the human homolog of the murine cycles (947C for 0 s; 587C for 3 s; 727C for 15 s). The primers MFG-E8 gene (7). The murine MFG-E8 cDNA sequence enused are huMFG-E8F1(5*-ggtgcatgtcaacctgtttgagac-3*) and codes a 463-amino-acid polypeptide with two epidermal huMFG-E8B1(5*-cagtccagttcgcacgtcattac-3*) located in the Cgrowth factor-like repeat domains linked to two 142-residue terminal region of the cDNA template (nucleotides 576– tandem repeated C-terminal domains. These latter domains 1002). Labeling was performed using a Rediprime random share 42% overall amino acid identity with the C1 and C2 primer labeling kit (Amersham) and unincorporated a[P]domains of human coagulation factors VIII and V (7) and dCTP was removed by using ProbeQuant G-50 microcolumns contain a 22-amino-acid sequence with over 80% homology (Pharmacia Biotech). Hybridization was performed for 24 h to a phosphatidylserine binding region in the human coagulafollowed by a posthybridization wash performed according to tion factors (3). Buse et al. (in preparation) have shown that the Genome Systems protocol; autoradiography was perMFG-E8 expression undergoes a 10to 20-fold induction in formed for 3 days. The single positive clone was ordered from mouse mammary epithelial cells concordant with the inducGenome Systems and confirmed by Southern blot hybridization of apoptosis in these same cells in the mammary gland tion with the same probe (data not shown). at the end of lactation. This induced apoptosis is critical for The huMFG-E8 PAC clone was localized on human target the normal process of involution, the developmental phase of chromosomes by fluorescence in situ hybridization (FISH) as breast tissue remodeling wherein epithelial cells are recogdescribed elsewhere (6). Briefly, DNA was extracted from an nized and ingested by infiltrating macrophages and the mamovernight culture using a modified alkaline lysis method (5). mary gland becomes repopulated with adipocytes and other The DNA was then labeled with digoxigenin–dUTP (Boehstromal cells as it returns to the pre-pregnant state. Buse et ringer-Mannheim) by nick-translation using the DNase-polyal. also show that MFG-E8 has phosphatidylserine binding merase mix from the BioNick Labeling System (BRL Life specificity and, at the onset of involution, is exposed as a Technology). Labeled probe was then hybridized to slides conperipheral membrane protein on the outer surface of epithetaining human metaphase chromosomes. Hybridized signal lial cells destined to be ingested by macrophages. Since movewas detected with FITC–anti-digoxigenin antibody (Boehment of phosphatidylserine from the inner to the outer leaflet ringer-Mannheim). The location of the hybridization signal of plasma membrane has been implicated as a signal for macon chromosome 15 band q25 was determined by DAPI bandrophage recognition of lymphocytes induced to enter ing and the fractional length measurement Flpter 0.86 was apoptosis (2), MFG-E8 has been postulated to be an inducible determined by digital imaging microscopy (8). The mapping marker that allows macrophage recognition of apoptotic epidata for the huMFG-E8 clone may be viewed at the Lawrence thelial cells. Berkeley National Laboratory, Resource for Molecular CytoThe human homolog of murine MFG-E8 was identified in genetics home page at http://www-rmc.lbl. gov. a human infant brain cDNA library (1) by a database search. This human cDNA clone has been sequenced (Wu and Parry, unpublished data) and contains coding sequence for a 387REFERENCES amino-acid polypeptide of which 263 (68%) are identical or