The "MAN antigens" are polypeptides recognized by autoantibodies from a patient with a collagen vascular disease and localized to the nuclear envelope. We now show that one of the human MAN antigens termed MAN1 is a 82.3-kDa protein with an amino-terminal domain followed by two hydrophobic segments and a carboxylterminal tail. The MAN1 gene contains seven proteincoding exons and is assigned to human chromosome 12q14. Its mRNA is approximately 5.5 kilobases and is detected in several different cell types that were examined. Cell extraction experiments show that MAN1 is an integral membrane protein. When expressed in transfected cells, MAN1 is exclusively targeted to the nuclear envelope, consistent with an inner nuclear membrane localization. Protein sequence analysis reveals that MAN1 shares a conserved globular domain of approximately 40 amino acids, which we term the LEM module, with inner nuclear membrane proteins lamina-associated polypeptide 2 and emerin. The LEM module is also present in two proteins of Caenorhabditis elegans. These results show that MAN1 is an integral protein of the inner nuclear membrane that shares the LEM module with other proteins of this subcellular localization.Only a few proteins other than the nuclear lamins, some with various isoforms, have been localized to the inner nuclear membrane during interphase. The first to be identified was avian lamin B receptor (LBR) 1 (1, 2). LBR was subsequently characterized in mammals (3-6), and an immunochemically crossreactive protein has been identified in sea urchins (7). LBR has a nucleoplasmic, amino-terminal domain of approximately 200 amino acids that binds to B-type lamins and chromatin proteins and confers inner nuclear membrane retention (2, 4, 8 -12). The amino-terminal domain of LBR is followed by a hydrophobic domain with eight putative transmembrane segments that is similar in sequence to sterol reductases, including two human proteins of the endoplasmic reticulum, one of which is a 7-dehydrocholesterol reductase (13).Two other integral proteins of the inner nuclear membrane have been termed lamina-associated polypeptides (LAPs). Three related isoforms of rat LAP1 (LAP1A, LAP1B, and LAP1C) were identified by reaction with a single monoclonal antibody and shown to be integral membrane proteins associated with the nuclear lamina (14). LAP1C has a nucleoplasmic amino-terminal domain followed by one transmembrane segment, and the other LAP1 isoforms are probably of similar overall structure, arising from the same gene by alternative RNA splicing (15). LAP2 was also first identified by reaction with a monoclonal antibody and shown to be an integral membrane protein that binds to nuclear lamins and chromatin (16). Several isoforms of LAP2, which have also been called thymopoietins because they were thought to possibly be thymocyte growth factors, are generated by alternative RNA splicing (17-21). Some of the LAP2 isoforms are integral proteins of the inner nuclear membrane, with nucleoplasmic amino-terminal domains and single transmembr...

The characterization of the human antiserum designated MAN has led to the identification of a subset of non-lamin proteins that are exclusively located at the nuclear periphery in all vertebrate cell types examined, from human to fish. Immunoreactive protein species were shown to comprise three major polypeptides of Mr 78000, 58000 and 40000. These antigens co-partitioned with the nuclear lamina during in situ isolation of nuclear matrices from lamin A/C-positive and -negative mammalian cells. Using double immunofluorescence, the spatial relationship of MAN antigens to type-A and type-B lamins was further examined throughout the cell cycle of lamin A/C-positive mammalian cells. In interphase HeLa and 3T3 cells, MAN antigens colocalized with both types of lamins at the periphery of the nucleus, but were absent from intranuclear foci of lamin B. As HeLa cells proceeded into mitosis, MAN antigens were seen to segregate from lamins A/C and coredistribute with lamin B. Lamins A/C disassembled during late prophase/early prometaphase and reassociated with chromatin in telophase/cytokinesis. In contrast, MAN antigens and lamin B dispersed late during prometaphase and reassembled on chromosomes in anaphase. Altogether, our data suggest that MAN antigens may play key functions in the maintenance of the structural integrity of the nuclear compartment in vertebrate cells.

A great majority of patients seeking preimplantation genetic diagnosis (PGD) are women >35 years of age. In addition to being carriers for single gene defects, these women also have a higher risk of having children with Down's syndrome (trisomy 21). For these patients, it would be advantageous if a diagnostic test for trisomy 21 was developed, which could be used in conjunction with tests for single gene defects. Here, we assessed the feasibility of developing an accurate genetic test for diagnosing trisomy 21 and the mutation causing spinal muscular atrophy (SMA) in single cells using multiplex fluorescence polymerase chain reaction (PCR). Single- and two-round PCR were developed using a combination of primers for the survival motor neuron (SMN) gene exons 7 and 8 and two chromosome 21 short tandem repeats (STRs), D21S226 and D21S11. After only 36 cycles, 88 and 68% of normal single cells were screened for SMA mutations and trisomy 21 respectively. In multiplex PCR using only two primers (SMN exon 7 and D21S11) instead of four, the efficiency of SMA diagnosis was increased to 93%. In the same reactions, the D21S11 alleles were detected in 83% of the normal single cells. Clinical applications of this assay should enable detection of those embryos that have inherited three heterozygous alleles and, therefore, benefit many PGD patients who are at an increased risk of Down's syndrome.

The characterization of the human antiserum designated MAN has led to the identification of a subset of non-lamin proteins that are exclusively located at the nuclear periphery in all vertebrate cell types examined, from human to fish. Immunoreactive protein species were shown to comprise three major polypeptides of Mr 78000, 58000 and 40000. These antigens co-partitioned with the nuclear lamina during in situ isolation of nuclear matrices from lamin A/C-positive and -negative mammalian cells. Using double immunofluorescence, the spatial relationship of MAN antigens to type-A and type-B lamins was further examined throughout the cell cycle of lamin A/C-positive mammalian cells. In interphase HeLa and 3T3 cells, MAN antigens colocalized with both types of lamins at the periphery of the nucleus, but were absent from intranuclear foci of lamin B. As HeLa cells proceeded into mitosis, MAN antigens were seen to segregate from lamins A/C and coredistribute with lamin B. Lamins A/C disassembled during late prophase/early prometaphase and reassociated with chromatin in telophase/cytokinesis. In contrast, MAN antigens and lamin B dispersed late during prometaphase and reassembled on chromosomes in anaphase. Altogether, our data suggest that MAN antigens may play key functions in the maintenance of the structural integrity of the nuclear compartment in vertebrate cells.