Further studies on hybrid cell-surface antigens associated with human chromosome 11.

A new human immunogenetic cell-surface activity associated with human chromosome 11 in the AL human-Chinese hamster ovary cell hybrid is described. Like a1, but not a2, it is present on the human erythrocyte. By mutagenesis and selection, specific, stable, variants of the AL hybrid have been prepared exhibiting various combinations of a1, a2, a3, and lactic dehydrogenase A activities. The antigens of the AL system can be demonstrated by the horseradish peroxidase system which offers a promising approach to scanning of tissue cells.     

Further studies on a hybrid cell-surface antigen associated with human chromosome 11 using a monoclonal antibody

A monoclonal antibody has been obtained that recognizes an antigen encoded by human chromosome 11. We present evidence that this monoclonal antibody recognizes the same or a similar antigenic activity as that previously called a1. Genetic information necessary for a1 expression and recognition by the monoclonal antibody both map to 11p13 leads to 11pter. Mutants that have lost a1 are no longer recognized by the monoclonal antibody. The macroglycolipid fraction of human erythrocyte membranes which contains the a1 antigenic activity is able to convert antigen-negative Chinese hamster ovary cells into cells which are killed by the monoclonal antibody plus complement.     

Human chromosome 11 carries at least four genes controlling expression of cell-surface antigens

We have mapped two new genes to chromosome 11 which control the cell-surface expression of two distinct antigens defined by monoclonal antibodies. One of the antigens has a general tissue distribution and is associated with a molecular complex of two polypeptides of 80,000 dalton and 40,000 dalton molecular weight. The second antigen has a restricted tissue distribution and is carried on a polypeptide of 100,000 daltons. We have used a combination of genetic and biochemical techniques to demonstrate that these new markers are distinct from the antigens defined by the monoclonal antibodies F10.44.2 and W6/34 which are also encoded by genes on chromosome 11. It is concluded that human chromosome 11 carries at least four distinct genes controlling cell-surface antigen expression.     

Physical mapping of the genes for three components of the mouse DNA replication complex: polymerase alpha to the X chromosome, primase p49 subunit to chromosome 10, and primase p58 subunit to chromosome 1

DNA polymerase alpha and primase are two key enzymatic components of the eukaryotic DNA replication complex. In situ hybridization of cloned cDNAs for mouse DNA polymerase alpha and for the two subunits of mouse primase has been utilized to physically map these genes in the mouse genome. The DNA polymerase alpha gene (Pola) was mapped to the mouse X chromosome in region C-D. The gene encoding the p58 subunit of primase (Prim2) was located to mouse chromosome 1 in region A5-B and the p49 subunit gene (Prim1) was found to be on mouse chromosome 10 in the distal part of band D that is close to the telomere. Current knowledge of mouse and human conserved chromosomal regions along with the findings presented here lead to predictions of where the genes for the DNA primase subunits may be found in the human genome: the p58 subunit gene may be on human chromosome 2 and the p49 subunit gene on human chromosome 12. The mapping of Pola to region C-D of the mouse X chromosome adds a new marker in a conserved region between the mouse X chromosome and region Xp21-22.1 of the human X chromosome.     

Isolation and mapping of 62 new RFLP markers on human chromosome 11.

To obtain new RFLP markers on human chromosome 11 for a high-resolution map, we constructed a cosmid library from a Chinese hamster x human somatic hybrid cell line that retains only human chromosome 11 in a Chinese hamster genomic background. A total of 3,500 cosmids were isolated by colony hybridization with labeled human genomic DNA. DNA was prepared from 130 of these cosmid clones and examined for RFLP. In 62 of them, polymorphism was detected with one or more enzymes; four RFLPs were VNTR systems. All polymorphic clones were assigned to one of 22 intervals obtained by mapping on a deletion panel of 15 somatic hybrid cell lines containing parts of chromosome 11; 11 clones were finely mapped by in situ hybridization. Although RFLP markers were scattered on the whole chromosome, they were found predominantly in the regions of R-banding. These DNA markers will contribute to fine mapping of genes causing inherited disorders and tumor-suppressor genes that reside on chromosome 11. Furthermore, as one-third of the cosmid clones revealed a band or bands in Chinese hamster DNA, indicating sequence conservation, this subset of clones may be useful for isolating biologically important genes on chromosome 11.     

A human cell-surface antigen defined by a monoclonal antibody and controlled by a gene on chromosome 12

A murine monoclonal antibody 602-29, subclass IgG1, that recognizes an antigenic determinant expressed by most human cells is described. Immunoprecipitation and sodium dodecyl sulfate-gel electrophoresis analysis indicate that the antigenic determinant is carried by a protein with an apparent molecular weight of 21,000. The antigen is expressed by human-mouse somatic cell hybrids, and analysis of segregants that have lost human chromosomes indicates that the gene controlling expression of the 602-29 antigen is on chromosome 12.     

Distribution of the major histocompatibility complex antigens on different cellular components of human liver

Monoclonal mouse antibodies to the "framework" determinants of the class I and II molecules of the major histocompatibility complex (MHC) were used to demonstrate the presence of the MHC antigens in human liver. First, the localization of these antigens was demonstrated from frozen section histology with indirect FITC immunofluorescence and the cell component(s) binding the mouse antibody were identified by rabbit marker antisera and indirect TRITC immunofluorescence. Second, the antigen expression on the cell surface was analyzed by the Staphylococcus aureus rosette method from cytological cell smears. All antibodies reacted with cells in the liver sinusoids, both with the Kupffer cells and at least partially with the sinusoidal endothelial cells. The same antisera reacted also with the bile duct cells, though weaker, and with some stromal cells in close proximity of the blood vessels. The vascular endothelial cells of hepatic artery, hepatic vein, and portal vein displayed no reaction. Thus human liver differs strikingly from, e.g., human kidney, where the vascular endothelial cells contain large amounts of MHC antigens on the cell surface. This difference may be one explanation to why liver allografts are less promptly rejected than renal allografts in man.     

Physical linkage of three CD3 genes on human chromosome 11.

T-cell antigen receptors are associated on T cell surfaces with a complex of proteins called CD3 (formerly T3). Human CD3 consists of at least four proteins, gamma, delta, epsilon and zeta, and all but the latter have been cloned as cDNA. Using standard cloning techniques, together with field inversion gel electrophoresis, we have demonstrated the physical linkage of three CD3 genes. The genes for CD3 gamma and CD3 delta are situated close together, about 1.6 kb apart, organized in a head-to-head orientation. The gene encoding CD3 gamma has been sequenced, and is split into seven exons spread over 9 kb of DNA. Like CD3 delta, CD3 gamma gene has an unusual promoter which lacks a TATA-box and potential Sp1 binding sites. The CD3 gamma-CD3 delta gene pair is within 300 kb of the CD3 epsilon gene, and therefore these genes form a tightly linked cluster in chromosome 11 band q23. The clustering of the CD3 genes may be significant in terms of their simultaneous activation during T-cell development.     

Deletion mapping of H-Y antigen to the long arm of the human Y chromosome

A gene encoding or controlling the expression of the H-Y transplantation antigen was previously mapped to the human Y chromosome. We now report the sublocalization of this gene on the long arm of the human Y chromosome. Eight patients with Y-chromosomal abnormalities were examined with a series of existing and new DNA markers for the Y chromosome. The resulting deletion map was correlated with H-Y antigen expression. We conclude that the H-Y antigen gene maps to a portion of deletion interval 6 that is identified by specific DNA markers.     

Genetic mapping of the human tryptophan hydroxylase gene on chromosome 11, using an intronic conformational polymorphism.

The identification of polymorphic alleles at loci coding for functional genes is crucial for genetic association and linkage studies. Since the tryptophan hydroxylase (TPH) gene codes for the rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, it would be advantageous to identify a polymorphism in this gene. By examining introns of the human TPH gene by PCR amplification and analysis by the single-strand conformational polymorphism (SSCP) technique, an SSCP was revealed with two alleles that occur with frequencies of .40 and .60 in unrelated Caucasians. DNAs from 24 informative CEPH families were typed for the TPH intron polymorphism and analyzed with respect to 10 linked markers on chromosome 11, between p13 and p15, with the result that TPH was placed between D11S151 and D11S134. This region contains loci for several important genes, including those for Beckwith-Wiedemann syndrome and tyrosine hydroxylase.     

Monoclonal antibody 53.6 recognizes a novel proliferation-associated antigen encoded on human chromosome 11

This paper describes the characterization of a novel cell surface antigen associated with proliferation. Previous work demonstrated that monoclonal antibody 53.6 reacted with every human cell line tested, as well as with subpopulations of normal bone marrow and peripheral blood lymphocytes. Mitogen stimulation of peripheral blood lymphocytes with phytohemagglutinin resulted in increased expression of the antigen recognized by 53.6. Immunoprecipitation of biosynthetically labeled KG-1A cell extracts with 53.6 revealed that the antigen is a nonglycosylated acidic protein of Mr 34,000. Analysis of mouse-human hybrid cell lines indicated that the structural gene for the antigen is encoded on chromosome 11. The antigen recognized by 53.6 is distinct from previously described cell surface antigens based on its distribution on activated cells and biochemical characteristics. These studies indicate that the 53.6 antigen is a novel proliferation-associated antigen, and may be useful in analyzing lymphocyte activation.     

Isolation, characterization, and regional mapping of microclones from a human chromosome 21 microdissection library.

Thirty-four unique-sequence microclones were isolated from a previously described microdissection library of human chromosome 21 and were regionally mapped using a cell hybrid mapping panel which consists of six cell hybrids and divides chromosome 21 into eight regions. The mapping results showed that the microclones were unevenly distributed along chromosome 21, with the majority of microclones located in the distal half portion of the long arm, between 21q21.3 and 21qter. The number of unique-sequence clones began to decrease significantly from 21q21.2 to centromere and extending to the short arm. This finding is consistent with those reported in other chromosome 21 libraries. Thus, it may be inferred that the proximal portion of the long arm of chromosome 21 contains higher proportions of repetitive sequences, rather than unique sequences or genes. The microclones were also characterized for insert size and were used to identify the corresponding genomic fragments generated by HindIII. In addition, we demonstrated that the microclones with short inserts can be efficiently used to identify YAC (yeast artificial chromosome) clones with large inserts, for increased genomic coverage for high-resolution physical mapping. We also used 200 unique-sequence microclones to screen a human liver cDNA library and identified two cDNA clones which were regionally assigned to the 21q21.3-q22.1 region. Thus, generation of unique-sequence microclones from chromosome 21 appears to be useful to isolate and regionally map many cDNA clones, among which will be candidate genes for important diseases on chromosome 21, including Down syndrome, Alzheimer disease, amyotrophic lateral sclerosis, and one form of epilepsy.     

Multipoint mapping studies of six loci on chromosome 11

The six loci, beta-globin (HBBC), parathyroid hormone (PTH), oncogene c-Ha-ras-1 (HRAS1), insulin (INS), calcitonin (CAL) and catalase (CAT) loci, have been mapped to 11p in the order: CAT-CAL-PTH-HBBC-(HRAS1-INS). The purpose of the current study was to examine the linkage relationships, especially the multipoint relationships of these loci in detail. In the 18 families studied, a significant sex difference in recombination was found for the HBBC: HRAS1 linkage with more recombination in the male parent than the female parent (22 vs. 2%). The results of the multipoint analyses provided further evidence for the order CAT-CAL-PTH-HBBC-(HRAS1-INS); however, the order of the last two tightly linked loci is still not clear.     

Isolation and regional mapping of DNA sequences unique to human chromosome 21.

To isolate DNA sequences unique to chromosome 21 we have used a recombinant-DNA library, constructed from a mouse-human somatic-cell hybrid line containing chromosome 21 as the only human chromosome. Individual recombinant phage containing human DNA inserts were identified by their hybridization to total human DNA sequences and by their failure to hybridize to total mouse DNA sequences. A repeat-free human DNA fragment was then subcloned from each of 14 such recombinant phage. An independent somatic-cell hybrid was used to assign all 14 subcloned fragments to chromosome 21. Thirteen of the fragments have been regionally mapped using a somatic-cell hybrid containing a human 21 translocation chromosome. Two probes map proximal to the 21q21.2 translocation breakpoint, and 11 probes map distal to this breakpoint, placing them in the region 21q21.2-21q22. One of seven probes used to screen for restriction-fragment-length polymorphisms recognized polymorphic DNA fragments when hybridized to genomic DNA from unrelated individuals. These 14 unique probes provide useful tools for studying the structure and function of human chromosome 21 as well as for investigating the molecular biology of Down syndrome.     

Isolation and regional mapping of NotI and EagI clones from human chromosome 21

NotI and EagI boundary libraries were constructed for human chromosome 21. One hundred forty-seven clones were isolated from the somatic cell hybrid 72532X-6 and localized using a hybrid mapping panel. After identification of those clones, which were isolated more than once, as well as those probes derived from a previously unrecognized integrated non-chromosome-21 fragment, 58 individual boundary clones (plus 2 additional NotI-EcoRI clones isolated from a flow-sorted library) were localized to 11 separate regions. The distribution of these probes is highly nonrandom, with 50% of the clones located in the distal band 21q22.3. Two probes, Not50 and Eag101, map to regions in the very proximal long arm which may contain the gene responsible for familial Alzheimer's disease (AD1), and Not50 would appear to be more proximal than D21S16 (E9). Twenty-eight probes map to the region between superoxide dismutase (SOD1) and the ETS2 oncogene, which appears to contain genes responsible for many of the phenotypic features of Down syndrome. Twenty clones contain (GT)n repeats, as determined by hybridization to a CA polymer, and should provide additional highly polymorphic probes. Closure of gaps in the physical linkage map of chromosome 21 should be facilitated by the isolation of these probes, as they identify many of the unmethylated CpG-rich islands that have hindered pulsed-field gel analysis. They will also be useful in identifying a set of genes in proximity to NotI and EagI restriction sites, as well as conserved DNA sequences for comparative mapping studies.     

Isolation, localization, and physical mapping of a highly polymorphic locus on human chromosome 11q13.

A highly polymorphic repetitive sequence, D11S533, was isolated by oligonucleotide hybridization from an arrayed chromosome 11q-specific cosmid library. The DNA sequence of this element was determined and found to consist of a repetitive degenerate hexanucleotide sequence [T(Pu)T(Pu)T(Pu)]n extending over 438 bp. Southern blot analysis demonstrated that this element is relatively unique in the human genome. This sequence can be detected by amplification using the polymerase chain reaction (PCR) with oligonucleotide primers complementary to unique sequences flanking the repetitive element. This sequence displays a high degree of polymorphism, and analysis of 15 individuals demonstrated at least 10 alleles ranging in size from 300 to 900 bp. Fluorescence in situ hybridization was used to localize this sequence to 11q13 (FLpter 0.60 +/- 0.02). Pulsed-field gel electrophoresis and the isolation of yeast artificial chromosomes established the long-range physical map surrounding the locus. Because various alleles of this polymorphic sequence can be easily detected by PCR amplification, this probe has potential usefulness in genetic linkage mapping as well as identity testing.