Bioautographic visualization of aminoacylase-1: assignment of the structural gene ACY-1 to chromosome 3 in man.

A bioautographic assay was developed for the visualization of aminoacylase-1 (N-acylamino acid aminohydrolase, ACY-1; EC 3.5.1.14) after zone electrophoresis. Bioautography and species differences in electrophoretic mobility of ACY-1 made it possible to investigate the chromosome assignment of the gene for human ACY-1 using human--mouse somatic cell hybrids. Human ACY-1 segregated concordantly with beta-galactosidase-A (beta GALA; EC 3.2.1.23) but showed discordant segregation with 32 other markers representing 23 linkage groups. The beta GALA gene has been previously assigned to chromosome 3. From this evidence and confirming chromosome analyses, ACY-1 has been assigned to chromosome 3. A genetic polymorphism in the electrophoretic mobility of ACY was observed in mouse strains, demonstrating that this enzyme can be mapped in genetic crosses of Mus musculus.     

Mapping of aminoacylase-1 and beta-galactosidase-A to homologous regions of human chromosome 3 and mouse chromosome 9 suggests location of additional genes.

Conserved linkage groups have been found on the X and autosomal chromosomes in several mammalian species. The identification of conserved chromosomal regions has potential for predicting gene location in mammals, particularly in humans. The genes for human aminoacylase-1 (ACY1, N-acylamino acid aminohydrolase, E.C.3.5.1.14), an enzyme in amino acid metabolism, and beta-galactosidase-A (GLB1, E.C.3.2.1.23), deficient in GM1-gangliosidosis, have been assigned to human chromosome 3. Using human-mouse somatic cell hybrids segregating translocations of human chromosome 3, expression of both ACY1 and GLB1 correlated with the presence of the p21 leads to q21 region of chromosome 3. In a previous study, assignment of these genes to mouse chromosome 9 used mouse-Chinese hamster somatic cell hybrids, eliminating mouse chromosomes. To approximate the size of the conserved region in the mouse, experiments were performed with recombinant inbred mouse strains. An electrophoretic variant of ACY-1 in mouse strains was used to map the Acy-1 gene 10.7 map U from the beta-galactosidase locus. These data suggest that there is a region of homology within the p21 leads to q21 region of human chromosome 3 and a segment of mouse chromosome 9. Since the mouse transferrin gene (Trf) is closely linked to the aminoacylase and beta-galactosidase loci, we predict that the human transferrin (TF) gene is on chromosome 3.     

Assignment of the structural gene encoding human aspartylglucosaminidase to the long arm of chromosome 4 (4q21----4qter)

The structural gene for the human lysosomal enzyme aspartylglucosaminidase (AGA) has been assigned to chromosome 4 using somatic cell hybridization techniques. The human monomeric enzyme was detected in Chinese hamster-human cell hybrids by a thermal denaturation assay that selectively inactivated the Chinese hamster isozyme, while the thermostable human enzyme retained activity. Twenty informative hybrid clones, derived from seven independent fusions, were analyzed for the presence of human AGA activity and their human chromosomal constitutions. Without exception, the presence of human AGA in these hybrids was correlated with the presence of human chromosome 4. All other human chromosomes were excluded by discordant segregation of the human enzyme and other chromosomes. Two hybrid clones, with interspecific Chinese hamster-human chromosome translocations involving the long arm of human chromosome 4, permitted the assignment of human AGA to the region 4q21----4qter.     

Gene mapping in Mus musculus by interspecific cell hybridization: assignment of the genes for tripeptidase-1 to chromosome 10, dipeptidase-2 to chromosome 18, acid phosphatase-1 to chromosome 12, and adenylate kinase-1 to chromosome 2.

Chinese hamster X mouse somatic cell hybrids segregating mouse chromosomes were examined for their mouse chromosome content using trypsin-Giemsa (GTG) banding and Hoechst 33258 staining techniques. Simultaneously, they were scored for the presence of 24 mouse enzymes. The results confirm the assignments of 11 genes previously mapped by sexual genetics: Dip-1 and Id-1 to chromosome 1; Pgm-2 and Pgd to 4; Pgm-1 to 5; Gpi-1 to 7; Gr-1 to 8; Mpi-1 and Mod-1 to 9; Np-1 and Es-10 to 14. They also confirm chromosomally the assignments of 3 genes that were made by other somatic cell genetic studies: Aprt to 8; Hprt and alpha-gal to the X chromosome. But most importantly, four enzyme loci are assigned to four chromosomes that until now were not known to carry a biochemical marker which is expressed in cultured cells: Trip-1 to 10; Dip-2 to 18; Acp-1 to 12; and Ak-1 to 2. Cytogenetic examination of clones showing discordant segregation of HPRT and A-GAL, suggested the assignment of alpha-gal to region XE leads to XF of the mouse X chromosome. The cytologic studies provide a comparison between data from sexual genetics and somatic cell hybrids and validate hybrid cell techniques. They provide evidence of the reliability of scoring chromosomes by GTG and Hoechst staining and stress the importance of identifying clones with multiple chromosome rearrangements. Striking examples of norandom segregation of mouse chromosomes were observed in these hybrids with preferential retention of 15 and segregation of 11 and the Y chromosome.     

Quantitative analysis of human chromosome segregation in man-mouse somatic cell hybrids.

The presumed random and independent process of human chromosome segregation in man-mouse somatic cell hybrids was studied. The results of chromosome analysis on 196 cells from 15 related hybrid strains have provided the first convincing evidence that segregation of human chromosomes can be nonindependent and often concordant. Different human chromosomes were not retained with equal frequency in these hybrid clones. Some were present in 80% of all the cells, whereas others appeared in less than 10% of the same cells. Linear regression analysis was used to test for correlation of the frequencies of all pair-wise combinations of human chromosomes present in these hybrid clones. Twenty-two of 136 possible correlations were statistically significant, indicating that concordant segregation of particular pairs of human chromosomes is a rather frequent occurrence.     

Localization of the human alpha-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay

We have used 16 human × mouse somatic cell hybrids containing a variable number of human chromosomes to demonstrate that the human α-globin gene is on chromosome 16. Globin gene sequences were detected by annealing purified human α-globin complementary DNA to DNA extracted from hybrid cells. Human and mouse chromosomes were distinguished by Hoechst fluorescent centromeric banding, and the individual human chromosomes were identified in the same spreads by Giemsa trypsin banding. Isozyme markers for 17 different human chromosomes were also tested in the 16 clones which have been characterized. The absence of chromosomal translocation in all hybrid clones strongly positive for the α-globin gene was established by differential staining of mouse and human chromosomes with Giemsa 11 staining. The presence of human chromosomes in hybrid cell clones which were devoid of human α-globin genes served to exclude all human chromosomes except 6, 9, 14 and 16. Among the clones negative for human α-globin sequences, one contained chromosome 2 (JFA 14a 5), three contained chromosome 4 (AHA 16E, AHA 3D and WAV R4D) and two contained chromosome 5 (AHA 16E and JFA14a 13 5) in >10% of metaphase spreads. These data excluded human chromosomes 2, 4 and 5 which had been suggested by other investigators to contain human globin genes. Only chromosome 16 was present in each one of the three hybrid cell clones found to be strongly positive for the human α-globin gene. Two clones (WAIV A and WAV) positive for the human α-globin gene and chromosome 16 were counter-selected in medium which kills cells retaining chromosome 16. In each case, the resulting hybrid populations lacked both human chromosome 16 and the α-globin gene. These studies establish the localization of the human α-globin gene to chromosome 16 and represent the first assignment of a nonexpressed unique gene by direct detection of its DNA sequences in somatic cell hybrids.     

Chromosome mapping of human cell surface molecules: monoclonal anti-human lymphocyte antibodies 4F2, A3D8, and A1G3 define antigens controlled by different regions of chromosome 11

Monoclonal antibodies 4F2, A3D8, and A1G3, directed against cell surface antigens present on subsets of human cells, were used to identify the human chromosome regions that code for the antigenic determinants. Human fibroblasts expressed all three antigens, and no cross-reactivity with Chinese hamster or mouse cells was found. Fourteen rodent X human somatic cell hybrids, derived from six different human donors and from two different Chinese hamster and one mouse cell line, were studied simultaneously for human chromosome content and for antibody binding as detected by indirect immunofluorescence. Concordancy with binding of all three antibodies was observed only for human chromosome 11. All other chromosomes were excluded by three or more discordant hybrid clones. Data from six hybrids containing three different regions of chromosome 11 indicate that it is the long arm of chromosome 11 which is both necessary and sufficient for expression of the human antigen defined by 4F2 while the antigen(s) defined by A3D8 and A1G3 map to short arm.     

X-Linkage of a Human Genetic Locus That Corrects the DNA Synthesis Lesion in tsC1AGOH Mouse Cells

GM 126 diploid fibroblasts were fused with a heat-sensitive mouse cell mutant defective in DNA synthesis, and primary hybrids were selected at permissive and nonpermissive temperatures in HAT medium. Primary hybrids, primary hybrid clones back-selected in 8-azaguanine at the permissive temperature, and subclones of heat-resistant primary hybrids isolated under nonselective conditions or after 8-azaguanine treatment were tested for heat sensitivity, the expression of 26 human enzymes assigned to 19 different human chromosomes, and the presence of human chromosomes. Only the human X chromosome and X-linked marker enzymes exhibited a clear pattern of concordant segregation with the heat-resistant phenotype. On the basis of these observations, we have defined the human genetic locus that corrects the heat-sensitive lesion in tsC1AGOH as hrC1AGOH and have assigned this locus to the X chromosome. This observation provides the first instance where two selectable markers (heat resistance and 8-azaguanine sensitivity) are found on a single human chromosome and suggests that these markers may prove to be a valuable push-pull selective system of use in determining the linear arrangement of genes on human chromosomes by somatic cell genetics.     

Stability of the "two active X" phenotype in triploid somatic cells.

We examined triploid cells of XXY karyotype heterozygous for glucose 6 phosphate dehydrogenase (G6PD) electrophoretic variants with regard to the stability of their X chromosome phenotype. Clonal populations of cells derived from these human fibroblasts maintained a precise 1:2:1 ratio of A:heteropolymer:B isozymes throughout their life span, indicating stability of the two active X chromosomes in these cells. To determine the influence of the autosomal complement on X chromosome expression, we attempted to perturb the relationship. Fusion of these triploid cells with human diploid fibroblasts carrying a novel G6PD variant (B') resulted in heterokaryons exprssing a novel heteropolymer, presumably indicating that all three parental X chromosomes were active. However, no derepression of the inactive X chromosome was observed. Analysis of interspecific hybrids derived from triploid cells and mouse fibroblasts confirmed that activity of parental X chromosomes is maintained. Some human mouse hybrid clones, however, expressed only a single human G6PD isozyme, probably attributable to segregation of the pertinent X chromosome, but elimination of a relevant autosome cannot be excluded. The triploid cells transformed by SV40 showed alterations in LDH pattern and an approximately 10-20% decrease in chromosome number, but maintained the precise G6PD phenotype of the untransformed cell. These studies provide evidence for the stability of the X chromosome phenotype in triploid cells.     

The gene coding for a sphingolipid activator protein,SAP-1, is on human chromosome 10

Summary SAP-1 is a sphingolipid activator protein found in human tissues required for the enzymatic hydrolysis of GM1 ganglioside and sulfatide. It appears to be missing in patients who have a genetic lipidosis resembling juvenile metachromatic leukodystrophy. Using rabbit antibodies against human SAP-1 it could be visualized in extracts from cultured human skin fibroblasts after sodium dodecylsulfate-polyacrylamide gel electrophoresis, followed by electroblotting to nitrocellulose membrane and immunochemical staining (Western blotting). A series of 23 human-Chinese hamster ovary cell hybrids containing different human chromosomes were examined. The parent Chinese hamster ovary cells did not have a reacting protein in the region of human SAP-1. Only in the eight hybrid clones containing human chromosome 10 was a reacting protein identified. Other chromosomes were excluded by this method. Therefore the gene for SAP-1 and the genetic mutation resulting in a fatal lipidosis are located on human chromosome 10. Present address: Department of Pediatrics, Osaka University Medical School, Fukushima-Ku, Osaka, Japan     

Regional localization of the genes for human HEXB. PGK, GALA. HPRT, G6PD by somatic cell hybridization

Twenty independent man-mouse (Cl1D,LA/TK-, HPRT-) and man-hamster (CH,HPRT-) hybrids using female human cells with balanced reciprocal translocation XX,t(X;5)(q21;q11) were analyzed for human genes localized on chromosome 5 (HEXB), on chromosome X (PGK, GALA, HPRT, G6PD) and for the different chromosomes in relation with the balanced reciprocal translocation (chr.5, chr.5q-, chr.Xq+, chr.X). The different results obtained indicate that the genes for human markers HEXB, PGK are on Xq+, and that the genes for human markers GALA, G6PD are on 5q-. These data implicate finally the following localizations: HEXB on 5q11 leads to 5qter; PGK on Xq21 leads to Xpter; GALA, HPRT, G6PD on Xq21 leads to Xqter.     

Human lysosomal genes: Arylsulfatase A and β-Galactosidase

The segregation of human lysosomal arylsulfatase A (ARS-A) has been evaluated in 50 primary hybrid clones derived from four separate fusions involving WBCs from two unrelated individuals and three hamster cell lines. ARS-A was expressed in the hybrids as a dimeric molecule of very similar or identical subunits. The expression of this enzyme was concordant with that of mitochondrial aconitase (ACON-M), an isozyme assigned to chromosome 22, in all 50 clones and with chromosome 22 segregation in all but one of the 29 karyotyped hybrids. No other human chromosome cosegregated with 22 in these clones, suggesting that this enzyme is specified in hybrid cells by a locus (or loci) on a single chromosome. beta-Galactosidase (B-GAL) expression was analyzed with two different electrophoresis systems and with a number of cell extract preparation methods in 39 of the primary hybrid clones. The B-GAL isozyme expressed in these hybrid cells was concordant with the expression of glutathione peroxidase-1 (GPX-1), an isozyme assigned to chromosome 3, in all 39 clones and with the segregation of this chromosome in 97% of the 29 karyotyped hybrids. These observations substantiate the prior tentative assignments of an ARS-A locus to chromosome 22 and a B-GAL locus to chromosome 3 (Bruns et al., 1978a, b). The implications of the chromosome assignments of loci for 12 human lysosomal enzymes for the cellular assembly of these organelles are discussed.     

Chromosome segregation from cell hybrids. VII. Reverse segregation from karyoplast hybrids suggests control by cytoplasmic factors.

Using human and Chinese hamster established lines as cell parents, we constructed hamster-human cell hybrids and human cell - hamster karyoplast hybrids. The cell hybrids retained one or two sets of hamster chromosomes and lost most of the human chromosomes. The karyoplast hybrids, however, retained a full set of human chromosomes and lost most of the Chinese hamster chromosomes. This reverse segregation pattern implies that cytoplasmic factors are major determinants of the direction of chromosome segregation.     

A gene on pig chromosome 14 suppresses cellular anchorage independence of the mouse cell line GM05267.

We have generated pig-mouse somatic cell hybrids by fusing normal pig fibroblasts with an anchorage independent mouse cell line GM05267. High quality G-banding analysis was applied to a set of 18 hybrid cell lines derived from 15 independent hybrids and chromosomes were identified. Cytogenetic analysis showed that all hybrids contained one or several pig chromosomes with normal morphology devoid of any structural changes. Out of 18 hybrids tested for colony formation in soft agar, 15 were suppressed for anchorage independence while the remaining three were not suppressed. Correlation of the cellular phenotype with the pig chromosome content of the hybrids suggests that the suppressor function for anchorage independence is located on pig chromosome (SSC) 14. We have previously shown that a suppressor gene for anchorage independence (SAI1) is located on rat chromosome (RNO) 5 and another suppressor gene for the same phenotype is located on human chromosome (HSA) 9. Given the genetic homology of both RNO5 and HSA9 with two pig chromosomes including SSC14, the third suppressor gene we have mapped on SSC14 may well be a functional homologue of the previously identified rat and human genes.     

Human cell surface antigens coded by genes on chromosome 21

Clones of man-mouse hybrids derived from four different crosses which retained a very limited number of human chromosomes were studied for the expression of human cell surface antigens. In testing a variety of rabbit antisera to human cells and tissues, it was found that an antiserum to Daudi cells recognizes human species-specific antigen(s) on three ‘WA’ clones, all of which carried human chromosome 21. Absorption of the antiserum with any of the clones abolished its activity against all clones, indicating that the antiserum recognized the same antigen(s) on these clones. The antigen(s) was shown to be present on normal human lymphocytes, more on B than on T cells, but apparently absent from erythrocytes. C3H mice, from which the murine parent originated, were immunized with the WA clones carrying human chromosome 21. The resultant antisera reacted with clones carrying chromosome 21 but not with clones which did not retain this chromosome, even though some of these clones possessed many other human chromosomes. The murine antisera reacted with some, but not all, human peripheral blood lymphocytes tested. Absorption studies clearly showed the multiple nature of the antigens recognized by these antisera. Studies on cells of identical twins provided evidence that these antigens are inheritable.     

Hexosaminidase isozyme in type O Gm2 gangliosidosis (Sandhoff-Jatzkewitz disease).

The residual enzyme of the fibroblasts of a child with homozygous type 0 GM2 gangliosidosis (Sandhoff-Jatzkewitz disease) has been found to correspond with a minor fraction of enzyme which can be isolated from normal fibroblasts by repeated chromatography. This enzyme is designated as hexosaminidase (hex) S. It reacts with antiserum prepared against homogeneous hex A but not with serum prepared against homogeneous hex B. These findings support our previously described model of the relationship between hex A and hex G: hex A has the structure (alpha beta)3, while hex B is (beta)6. Type B GM2 gangliosidosis (Tay-Sachs disease) is the alpha- mutation, while type 0 GM2 gangliosidosis (Sandhoff-Jatzkewitz disease) is the beta- mutation. In the absence of normal beta subunits there is increased polymerization of alpha subunits forming hex S, which probably has a structure of (alpha)6. A parallel between the thalassemias and GM2 gangliosidosis is evident: deficiency of one of the chains of which the protein is composed leads to an excess of polymers comprised of the other chains. In type B GM2 gangliosidosis, the excess of beta chanis leads to increased amounts of hex B beta)6; in type 0 GM2 gangliosidosis, the excess of alpha chains leads to formation of increased amounts of the alpha chain polymer, hex S.     

Assignment of the gene governing cellular ouabain resistance to Mus musculus chromosome 3 using human/mouse microcell hybrids

Human/mouse microcell hybrids were used to establish the assignment of the gene governing resistance to the cardiac glycoside ouabain (Oua-1) to Mus musculus chromosome 3. Microcells were prepared from primary mouse embryo fibroblasts and fused with HeLa S3 cells, and microcell hybrids were isolated and maintained in medium containing 10^–6 m ouabain. Resistance to ouabain was not expressed concordantly with any of 26 murine isozyme markers. Karyotypic analysis of five primary clones showed that one to five murine chromosomes had been transferred from donor to recipient in these experiments. Only mouse chromosome 3 was common to all ouabain-resistant primary clones. Both ouabain-resistant and -sensitive subclones were isolated from hybrids grown in the absence of selective pressure, and karyotyping showed that loss of resistance to ouabain was concordant with the loss of murine chromosome 3.These studies were supported by Grant GM9966 from the National Institutes of Health.     

Chromosomal localization of the human histamine H1-receptor gene

We have assigned the human histamine H₁-receptor gene to chromosome 3 by Southern blot analysis of a chromosome mapping panel constructed from humanhamster somatic cell hybrids. This assignment was confirmed by in situ hybridization on metaphase chromosomes and involved bands 3p14–p21.     

Introduction of new genetic markers on human chromosomes

The purpose of this study was to use DNA transfection and microcell chromosome transfer techniques to engineer a human chromosome containing multiple biochemical markers for which selectable growth conditions exist. The starting chromosome was a t(X;3)(3pter----3p12::Xq26----Xpter) chromosome from a reciprocal translocation in the normal human fibroblast cell line GM0439. This chromosome was transferred to a HPRT (hypoxanthine phosphoribosyltransferase)-deficient mouse A9 cell line by microcell fusion and selected under growth conditions (HAT medium) for the HPRT gene on the human t(X;3) chromosome. A resultant HAT-resistant cell line (A9(GM0439)-1) contained a single human t(X;3) chromosome. In order to introduce a second selectable genetic marker to the t(X;3) chromosome, A9(GM0439)-1 cells were transfected with pcDneo plasmid DNA. Colonies resistant to both G418 and HAT medium (G418r/HATr) were selected. To obtain A9 cells that contained a t(X;3) chromosome with an integrated neo gene, the microcell transfer step was repeated and doubly resistant cells were selected. G418r/HATr colonies arose at a frequently of 0.09 to 0.23 x 10(-6) per recipient cell. Of seven primary microcell hybrid clones, four yielded G418r/HATr clones at a detectable frequency (0.09 to 3.4 x 10(-6)) after a second round of microcell transfer. Doubly resistant cells were not observed after microcell chromosome transfers from three clones, presumably because the markers were on different chromosomes. The secondary G418r/HATr microcell hybrids contained at least one copy of the human t(X;3) chromosome and in situ hybridization with one of these clones confirmed the presence of a neo-tagged t(X;3) human chromosome. These results demonstrate that microcell chromosome transfer can be used to select chromosomes containing multiple markers.     

Epstein-Barr virus in somatic cell hybrids between mouse cells and human nasopharyngeal carcinoma cells.

Somatic cell hybrids between mouse cells and cells derived directly from NPC biopsies were produced in order to study the association of the Epstein-Barr virus (EBV) genome and the expression of Epstein-Barr nuclear antigen (EBNA) with the human chromosome(s). All attempts to correlate the presence of EBV-DNA and the expression of EBNA with the presence of a particular human chromosome(s) showed that the segregation of EBV-DNA or of EBNA and human chromosomes was dysconcordant. The data, therefore, suggest that in the hybrids studied the presence of EBA-DNA is not determined by the presence of a specific human chromosome.