The Gene for the Leu-M2 (Mac-120) Antigen is Located on Human Chromosome 11

Leu-M2 (Mac-120) antigen is a cell-surface marker present on a subpopulation of human blood monocytes and platelets. Its expression on monocytes and macrophages is necessary for their ability to present antigen to T lymphocytes. Only Leu-M2+ cells display the human Ia-like determinant, HLA-DS, which seems important in antigen presentation and stimulation in the autologous MLR. Expression of the Leu-M2/Mac-120 antigen was studied by indirect immunofluorescence analysis and flow cytofluorometry in mouse-human somatic cell hybrids segregating human chromosomes. In the mouse-human hybrid clones, expression of the Leu-M2/Mac-120 antigen was dependent on the presence of human chromosome 11. This was verified by fluorescence-activated cell sorting of heterogeneous hybrid cell populations into antigen-positive and-negative fractions. The Leu-M2 antigen co-segregated with chromosome 11. The gene controlling the expression of the Leu-M2/Mac-120 antigen thus is assigned to human chromosome 11.     

Human muscle cell surface antigen 16.3A5 is encoded by a gene on chromosome 11

A monoclonal antibody (McAb), 16.3A5, has been characterized that reacts with a cell surface antigen of human muscle cells and a variety of nonmuscle cells. The gene controlling synthesis of 16.3AS antigen has been assigned to human chromosome 11 by assessing McAb reactivity on a panel of mouse-human cell hybrids. The 16.3A5 has a novel specificity distinct from other chromosome 11-encoded antigens such as W6/34, F10.44.2, TRA1.10, and 4D12 antigens.     

Genes for human vitamin K-dependent plasma proteins C and S are located on chromosomes 2 and 3, respectively

cDNAs encoding human proteins C and S have been used to screen a panel of mouse-human somatic cell hybrids to determine the chromosomal location of their respective genes. The gene for human protein C is located on chromosome 2, whereas that for protein S is located on chromosome 3. Analysis of human genomic DNA restriction endonuclease fragmentation patterns suggests that the human protein S gene is greater than 40 kb in size and contains a minimum of 11 introns.     

Assignment of human nerve growth factor receptor gene to chromosome 17 and regulation of receptor expression in somatic cell hybrids

Nerve growth factor (NGF) is a polypeptide hormone which plays a central role in the development and growth of sympathetic and sensory neurons. The effects of NGF on target cells are mediated by a specific cell surface structure, nerve growth factor receptor (NGFr), which has been identified in human cells as a 75,000-mol-wt glycoprotein. We have used a monoclonal antibody to human NGFr to study cell-surface expression of the receptor on a panel of mouse-human neuroblastoma hybrids, and the serological typing results permit assignment of the gene coding for NGFr(NGFR) to chromosome 17q21-qter. In addition to mouse-human neuroblastoma hybrids, human NGFr was also detected on hybrids derived from fusions between mouse L-cell fibroblasts and human neuroblastoma and melanoma cells. Furthermore, induction of human NGFr expression was observed in hybrids derived from NGFr^– human kidney epithelial cells and mouse L cells, but not in hybrids derived from human kidney epithelial cells and mouse RAG kidney carcinoma cells. These results suggest that cell-surface expression of human NGFr is controlled by transacting regulatory signals.     

Assignment of gene for human cell-surface membrane antigen Trop-4 to chromosome 11

The gene (named MFI6) for a surface membrane antigen, Trop-4, is assigned to human chromosome 11 on the basis of studies using a mouse monoclonal antibody, immunofluorescence, fluorescence-activated cell sorting (FACS), immunoprecipitation, and mouse-human lymphocyte hybrids. The Trop-4 antigen is present on all human cell lines tested, on peripheral blood monocytes and granulocytes, and on a small fraction of peripheral blood lymphocytes, but is absent from erythrocytes. The Trop-4 monoclonal antibody precipitates an 85,000-dalton glycopolypeptide from hybrid cells containing human chromosome 11. However, in a human cell line expressing this antigen, a larger-molecular-weight species, 100–105,000 daltons was coprecipitated with the 85,000-dalton glycopeptide, and under nonreducing conditions a larger compound of 110–125,000 daltons was obtained. Although the Trop-4 antigen is of similar molecular weight to the Mab-4 and F10.44.2 antigens previously assigned to chromosome 11, it is shown to be different from them.     

The gene for human lymphocyte homing receptor is located on chromosome 11

The mouse monoclonal antibody Hermes-3 recognizes the human lymphocyte homing receptor. A panel of mouse-human T lymphocyte hybrids, carrying all mouse chromosomes and a limited number of human chromosomes, was analyzed for expression of human homing receptor by indirect immunofluorescence and immunoprecipitation of radiolabeled cell lysates with Hermes-3 antibody. Karyotypic analysis of the tested clones showed that the expression of human homing receptor correlated to the presence of human chromosome 11 in all but one clone. However, concanavalin A induced a weak to moderate expression of the homing receptor in this clone, but not in a chromosome 11- clone. Another clone, heterogeneous for the expression of homing receptor, was separated into a Hermes-3+ and a Hermes-3- fraction with a fluorescence-activated cell sorter. Karyotypic analysis performed after sorting showed human chromosome 11 to segregate with the Hermes-3 antigen. To confirm these data we correlated the expression of two chromosome 11-coded antigens, Trop-4 and Leu-7, with the expression of the homing receptor. In our hybrid clones these three antigens were expressed concordantly. The gene coding for the human lymphocyte homing receptor recognized by Hermes-3 is thus assigned to chromosome 11.     

Identification of 200,000-dalton human cell surface protein encoded by gene mapped to long arm of chromosome 11

Cell surface proteins and glycoproteins of human and Chinese hamster cells and their hybrid cell clones were analyzed by two-dimensional gel electrophoresis. The J1 clone of human-Chinese hamster hybrid cells contained chromosome 11 as its only human chromosome. The J1 cells expressed a glycoprotein of 200,000 daltons which was shared by human fibroblasts but not by the parental Chinese hamster ovary cells. The 200,000-dalton protein was identified as a cell surface protein by the method of lactoperoxidase-catalyzed iodination. The protein was electrophoretically purified from radioiodinated cultures of human fibroblasts and J1 cells and subjected to the analysis of tryptic peptides by thin-layer electrophoresis followed by chromatography. The protein from both sources gave rise to fingerprints which closely resembled to each other. The results are consistent with a hypothesis that the 200,000-dalton protein of the J1 clone is of human origin. Analysis of segregant clones of J1 cells, which have deletions on human chromosome 11, has further suggested that the gene for this glycoprotein maps to the long arm of chromosome 11. A gene coding for the 200,000-dalton protein has not been previously mapped to this chromosome.     

Isolation of genomic clones homologous to transcribed sequences from human X chromosome

Several X chromosome DNA clones homologous to transcribed sequences were isolated from a human X chromosome library. The clones were selected for their ability to hybridize either with³²P -labeled human cDNA in the presence of an excess of unlabeled human repetitive DNA or with mouse fibroblast cDNA. The X chromosome specificity of these sequences was demonstrated by two criteria: A dosage effect was seen when the clones were hybridized to Southern blots of DNA from 1X and 5X cells, and they hybridized to DNA from mouse-human hybrid cells containing only the human X chromosome. The presence of transcribed sequences in these X clones was detected by hybridization with mouse cDNA or with human cDNA in the presence of unlabeled human repetitive sequences, by identifying restriction fragments which hybridize with cDNA but not with human repetitive DNA, and by hybridization with poly A⁺ RNA on Northern blots. These clones were mapped on the human X chromosomes using a panel of mouse-human somatic cell hybrids carrying various translocated human X chromosomes.     

Localization of gene for human syndecan,an integral membrane proteoglycan and a matrix receptor,to chromosome 2

Syndecan, a cell surface proteoglycan, is an integral membrane protein acting as a receptor for the extracellular matrix. For chromosomal localization of the human syndecan gene, a panel of mouse-human somatic cell hybrids was analyzed by Southern blotting using the cDNA probe for human syndecan. The hybrids were karyotyped at the time of DNA extraction. A band corresponding to the human syndecan gene in Southern blots was found only in a hybrid cell line containing human chromosome 2. This hybrid was subcloned and its subclones were analyzed by Southern blotting and karyotyped. Subclones carrying human chromosome 2 contained the syndecan gene, while subclones not carrying this chromosome did not. The human syndecan gene is thus assigned to chromosome 2.     

Three human cell surface antigen systems determined by genes on chromosome 12

The mouse monoclonal antibodies AbA123, AbA127, AbK152, AbM68, and AbV1 were derived after immunization with cultured human tumor cells or melanocytes. Antibodies AbA123, AbA127, and AbK152 recognize human cell surface antigens expressed on most cultured human cells and show an identical pattern when tested on a panel of 47 human cell lines. They recognize at least two different epitopes on the same glycoprotein complex, designated A123/A127, which consists of 30,000- and 40,000-mol-wt glycopeptides. Antigens M68 and V1 are also expressed on most cultured human cell types but show distinct patterns of distribution on the cell line panel. The antigens defined by AbM68 and AbV1 have the characteristics of glycolipids. They are heat stable, and immunoprecipitation of metabolically labeled cell lysates did not yield any detectable components when analyzed by SDS-polyacrylamide gel electrophoresis. Serological typing of a panel of 23 independently derived mouse-human and Chinese hamster-human somatic cell hybrids showed unequivocally that the expression of cell surface antigens A123/A127, M68, and V1 segregates with human chromosome 12. The analysis of hybrids containing karyotypically defined deletions of chromosome 12 permitted the assignment of the loci determining the expression of antigens A123/A127 and V1 to region 12cen-qter, and the locus determining the expression of antigen M68 to region 12cen-pter. These antigens can be distinguished from the cell surface molecules previously assigned to chromosome 12 and thus represent new assignments to this chromosome.     

Immunoglobulin heavy chain genes in humans are located on chromosome 14

The human immunoglobulin heavy chain gene complex has been assigned to chromosome 14 by filter hybridization of restriction digests of mouse-human somatic cell hybrids. Cloned DNA probes for both variable and constant regions were used.     

Localization of the murine leukemia inhibitory factor gene near the centromere on chromosome 11

Leukemia inhibitory factor (LIF) is a glycoprotein with divergent activities: It induces the differentiation of certain myeloid leukemic cells, inhibits the differentiation of embryonic stem cells, and promotes bone remodelling in vivo and in vitro. The murine LIF gene has been assigned to the proximal region of chromosome 11 at sub-bands A1-A2, by analysis of a panel of mouse x Chinese hamster somatic cell hybrids and by in situ hybridization. Interestingly, the proximal portion of chromosome 11 has been shown, by virtue of its parental origin effects, to contain gene(s) involved in fetal growth. It is also interesting that there is a preponderance of chromosome 11 abnormalities in embryonal carcinoma cells. The localization of the murine LIF gene confirms the homology of a portion of murine chromosome 11 with human chromosome 22q, the site of the human LIF gene.     

Integration of a dominant selectable marker into human chromosomes and transfer of marked chromosomes to mouse cells by microcell fusion

A method for the production of stable mouse-human cell hybrids containing a single human chromosome is described. As a first step in this method, a cloned selectable marker, the E. coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, was transferred to human cells to generate cell lines each carrying Ecogpt integrated into a different site. Human chromosomes marked with Ecogpt were transferred further into mouse cells by microcell fusion. Monochromosomal hybrids, in which the human chromosome is maintained by selection, have been produced for chromosomes 2, 5, 16, and a rearranged chromosome involving a translocation between chromosomes 1 and 2. In addition to these monochromosomal hybrids, we have also obtained monochromosomal hybrids for human chromosomes 6, 12, and 17 by selection for the loss of marked chromosome from the microcell hybrids each containing two human chromosomes. Although the human chromosome present in these hybrids cannot be maintained by selection, 80–90% of cells retained the transferred chromosome on continuous growth for 15 days. Monochromosomal hybrids would provide biological materials to construct genetic maps of human chromosomes. In addition, chromosomes marked with dominant selectable markers can be transferred further to any cell line of interest in inter- or intra-species combination.     

A hamster-human subchromosomal hybrid cell panel for chromosome 2

We have constructed hamster-human hybrid cell lines containing fragments of human chromosome 2 as their only source of human DNA. Microcell-mediated chromosome transfer was used to transfer human chromosome 2 from a monochromosomal mouse-human hybrid line to a radiation-sensitive hamster mutant (XR-V15B) defective in double-strand break rejoining. The human chromosome 2 carried theEcogpt gene and hybrids were selected using this marker. The transferred human chromosome was frequently broken, and the resulting microcell hybrids contained different sized segments of the q arm of chromosome 2. Two microcell hybrids were irradiated and fused to XR-V15B to generate additional hybrids bearing reduced amounts of human DNA. All hybrids were analyzed by PCR using primers specific for 27 human genes located on chromosome 2. From these data we have localized the integratedgpt gene on the human chromosome 2 to the region q36–37 and present a gene order for chromosome 2 markers.     

Assignment of gene(s) coding for antigen defined by monoclonal antibody 2B2

A mouse monoclonal antibody (2B2) recognizes an antigen which is present on most human peripheral blood leukocytes but is absent from most proliferating cells. The antibody precipitated two surface-labeled membrane glycopolypeptides with molecular weights of 86,000 and 145,000, and it was strongly mitogenic to normal human lymphocytes. Somatic cell hybrids have been used for assigning the genes coding for these membrane glycoproteins to human chromosome 21. The assignment was based on correlation of antigen expression on mouse-human T-lymphocyte hybrids with the presence of human chromosomes in the same hybrid clones.     

Genetic assignment of GP90, leukocyte adhesion glycoprotein to human chromosome 21

Intercellular adhesion among human leukocytes involves a cell-surface glycoprotein with an apparent molecular weight of 90,000 which forms complexes with higher-molecular-weight glycoproteins. A monoclonal antibody (60.3) against this glycoprotein blocks induced adhesion. Here we have shown that the antibody reacts with cell clones carrying human chromosome 21 in lymphocyte hybrids between an AKR mouse thymoma (BW5147) and human concanavalin A-activated peripheral blood lymphocytes. Cell sorting by FACS of a hybrid clone heterogeneous in the expression of the antigen identified by the 60.3 antibody yielded a positive fraction expressing the antigen and carrying human chromosome 21, and a negative fraction lacking both the antigen and chromosome 21. The gene coding for the cell adhesion glycoprotein is thus provisionally assigned to chromosome 21.     

Localization of the Murine Leukemia Inhibitory Factor Gene Near the Centromere on Chromosome 11

Abstract

Leukemia inhibitory factor (LIF) is a glycoprotein with divergent activities: It induces the differentiation of certain myeloid leukemic cells, inhibits the differentiation of embryonic stem cells, and promotes bone remodelling in vivo and in vitro. The murine LIF gene has been assigned to the proximal region of chromosome 11 at sub-bands A1-A2, by analysis of a panel of mouse x Chinese hamster somatic cell hybrids and by in situ hybridization. Interestingly, the proximal portion of chromosome 11 has been shown, by virtue of its parental origin effects, to contain gene(s) involved in fetal growth. It is also interesting that there is a preponderance of chromosome 11 abnormalities in embryonal carcinoma cells. The localization of the murine LIF gene confirms the homology of a portion of murine chromosome 11 with human chromosome 22q, the site of the human LIF gene.     

Development of a mouse model for HIV/AIDS

A small animal model would be very valuable for HIV/AIDS vaccine testing, investigating HIV pathophysiology, and exploring anti-HIV therapeutics. Unfortunately, HIV does not replicate in mouse cells. Provision of mouse cells with human CD4, CCR5 and cyclin T1 (cycT1) has uncovered a block to HIV assembly or release. Since mouse-human cell fusions allow viral replication, mouse cells lack at least one critical factor that permits completion of the viral life cycle. To identify this factor(s) we are employing 2 similar genetic approaches. Each cell line of a panel of monochromosomal mouse-human somatic cell hybrids was individually transduced with an HIV vector encoding both cycT1 and blasticidin resistance (HIV-CIB). Each was then transfected with vesicular stomatitis virus (VSV) G protein and measurable virus was recovered from only the hybrid-containing chromosome 2. This was verified with an M-tropic envelope and was shown to be specific to HIV. In addition, the amount of p24 release from that hybrid was substantially greater than that from the parent. A second cell line expressing chromosome 2 had a similar phenotype. CycT1 has been introduced into one chromosome 2 line to monitor the spread of HIV. In a related but separate approach, an entire collection of approximately 500 mouse-human microcell hybrids was transduced with HIV-CIB and broken down into manageable pools. Virus was similarly recovered as above from a few of the pools. Those pools were then broken down to clones and several cell clones have been identified that allow virus release. Revertants that no longer have the human chromosome are now being tested for loss of phenotype. Clones will then be tested for ability to support both HIV replication and Gag processing. Human chromosomal content of the clones of greatest interest will be determined by STS content analysis. Results from the 2 approaches are expected to be in agreement and may provide direction for an expression cloning approach.     

Pseudogene of dihydrolipoyl succinyltransferase (E2k) found by PCR amplification and direct sequencing of rodent-human cell hybrid DNAs

Previous studies have indicated that the cDNA for the E2k component of the human -ketoglutarate dehydrogenase complex (KGDHC) hybridized not only to a major locus on chromosome 14q24.3 in a region associated with familial Alzheimer's disease and with Joseph-Machado disease, but also to another locus on chromosome 1p31. We now report that PCR of genomic DNA and direct sequencing indicated that the chromosome 1 locus is an intronless pseudogene. PCR of genomic DNA amplified E2k fragments from mouse-human cell hybrids containing human chromosome 1 DNA but not from hybrids containing human chromosome 14 DNA. The resulting amplicons were of comparable sizes to those when the cDNA was used as template. The direct sequencing of these amplicons confirmed the lack of introns and indicated a frame shift, which led to the presence of four termination codons early in the coding region. PCR followed by direct sequencing of the amplicons appears to be a convenient method for identifying intronless pseudogenes.