Man-mouse Somatic Cell Hybrids Research Articles

Regional assignment of the human acid sphingomyelinase gene (SMPD1) by PCR analysis of somatic cell hybrids and in situ hybridization to 11p15.1→p15.4

Human acid sphingomyelinase (SMPD1) is the lysosomal phosphodiesterase that cleaves sphingomyelin to ceramide and phosphocholine. The deficient activity of SMPD1 is the enzymatic defect in Types A and B Niemann-Pick disease. Previously, the gene encoding human SMPD1 was assigned to chromosome 17 by the differential thermostability of human and hamster SMPD1 in somatic cell hybrids. The recent isolation of the human SMPD1 cDNA ( L. E. Quintern, E. H. Schuchman, O. Levran, M. Suchi, K. Ferlinz, H. Reinke, K. Sandhoff, and R. J. Desnick, 1989, EMBO J. 8: 2469–2473) permitted the mapping of this gene by molecular techniques. Oligonucleotide primers were synthesized to PCR amplify the human, but not murine, SMPD1 sequences in man-mouse somatic cell hybrids. In a panel of 15 hybrid cell lines, amplification of the human SMPD1 sequence was 100% concordant with the presence of human chromosome 11. For each of the other human chromosomes there were at least 6 discordant hybrid lines. Further analysis of somatic cell hybrids containing only chromosome 11 or chromosome 11 rearrangements localized the human SMPD1 gene to the region 11p15.1→p15.4. To provide an independent regional gene assignment, in situ hybridization was performed using the radiolabeled human SMPD1 cDNA. In the 58 metaphase cells examined, 34% of the 122 hybridization sites scored were located in the distal end of chromosome 11 with the major peak of hybridization at band 11p15. The absence of any other in situ hybridization site indicated the absence of pseudogenes or homologous sequences elsewhere in the genome. In contrast to the previous provisional localization to chromosome 17, these results assign a single locus for human SMPD1 to 11p15.1→p15.4.

The chromosomal localization of human beta-galactosidase revisited: a locus for beta-galactosidase on human chromosome 3 and for its protective protein on human chromosome 22.

A series of man-Chinese hamster and man-mouse somatic cell hybrids was investigated to study the localization of the genes coding for the human lysosomal enzyme beta-galactosidase (EC 3.2.1.23) and for its protective protein. Using a monoclonal antibody, raised against human placental beta-galactosidase, it was observed that the structural locus for the beta-galactosidase polypeptide is located on chromosome 3. The nature of the involvement of chromosome 22 in the expression of human beta-galactosidase was elucidated by metabolic labelling of the hybrids with radioactive amino acids, immunoprecipitation with monoclonal and polyclonal antibodies against beta-galactosidase, followed by analysis via gel electrophoresis and fluorography. The data show that the presence of chromosome 22 coincides with the presence of a 32 kd protein. This polypeptide, the "protective protein" was previously shown to be intimately associated with human beta-galactosidase. In addition, the protective protein was found to be essential for the in vivo stability of beta-galactosidase by aggregating beta-galactosidase monomers into high molecular weight multimers. Both chromosome 3 and 22 are therefore necessary to obtain normal levels of beta-galactosidase activity in human cells.

A Sequence Homologous to Rous Sarcoma Virus v-src is on Human Chromosome 20

Publisher Summary Cellular sequences (c- src ) homologous to the v- src gene of Rous sarcoma virus (RSV) are present in the genomic DNAs of all vertebrates examined and have been evolutionarily conserved. The v- src gene is responsible for the malignant transformation of cells and is expressed as a 60,000-dalton phosphoprotein, designated as pp6Ov-src, with protein kinase activity. Using Southern hybridization techniques and man-mouse somatic cell hybrids, a human c- src gene is assigned to chromosome 20 in the chapter. Cellular DNAs were cut with EcoRI and probed with a 3.1-kb Eco RI DNA fragment containing v- src derived from pSRA-2, a recombinant plasmid containing the genome of the Schmidt–Ruppin A strain of RSV. The v- src gene hybridizes principally to a large Eco RI fragment of human DNA and to two Eco RI fragments of mouse DNA. The presence of additional minor bands that hybridize to v- src suggests that other src -related genes might be present in human and mouse genomes. Human cells contain two related but distinct forms of pp60c-src; however, it is not known whether the two forms are the products of separate c- src genes.

Assignment of a fibronection gene to human chromosome 2 using monoclonal antibodies

The locus coding for the presumed structural gene for fibronectin has been mapped to human chromosome 2 using human-mouse somatic cell hybrids. The assignment of fibronectin has been made by testing man-mouse somatic cell hybrids with two anti-human fibronectin monoclonal antibodies which recognize different antigenic determinants of human, but not mouse, fibronectin, Both monoclonal antibodies demonstrate a highly concordant association between the presence of two different human fibronectin antigens and human chromosome 2.

Gene for glutathione S-transferase-1 (GST1) is on human chromosome 11.

The glutathione S-transferases (GST) are a group of related enzymes that can detoxify potentially carcinogenic electrophiles by conjugating them with reduced glutathione (GSH). The chromosomal location of one of the enzyme forms, GST1, reported recently to be polymorphic, was determined utilizing man-mouse somatic cell hybrids segregating human chromosomes. The expression of GST1 by hybrid clones was compared with that of 34 enzyme markers representing 23 chromosomes, and karyotypes of selected cell hybrids were analyzed. The evidence indicated that GST1 is assigned to chromosome 11 in humans. Utilizing and X/11 translocation segregating in hybrids, GST1 was localized to the p13 leads to qter region of chromosome 11.

Sequential staining with Hoechst 33258 and quinacrine mustard for the identification of human chromosomes in somatic cell hybrids

We have analysed metaphase chromosomes of man-mouse somatic cell hybrids using a sequential staining procedure involving the fluorescent DNA-binding stains, Hoechst 33258 and quinacrine mustard. This was found to be a simple and reliable means of differentiating the chromosomes of the two species and of identifying specific human chromosomes. In addition, this method will permit the study of the segregation of human chromosome homologues that are discordant for quinacrine mustard fluorescent polymorphisms.

Human beta-galactosidase and alpha-neuraminidase deficient mucolipidosis: genetic complementation analysis of the neuraminidase deficiency.

Human beta-galactosidase and alpha-neuraminidase deficient mucolipidosis [ML(gal-neur-)] is an inherited lysosomal enzymopathy which recently was designated as a sialidosis. We analyzed the neuraminidase deficiency of this disorder with genetic complementation analyses using a heterokaryon enrichment procedure. The genetic defects of two apparent variants of this disorder complemented the defects of the neuraminidase deficiency diseases, sialidosis I and mucolipidosis I, resulting in the restoration of neuraminidase activity in heterokaryons. The neuraminidase deficiency, therefore, may not be the primary defect in ML(gal-neur-) and is not an appropriate test for determining carrier status. The clinical and biochemical characteristics of this disorder suggest that a post-translational or processing event for these enzymes may be defective. The defect, however, is different from I-cell disease and pseudo-Hurler polydystrophy, two disorders of post-translational lysosomal enzyme biosynthesis, since complementation studies demonstrated recovery of intracellular beta-galactosidase and alpha-neuraminidase levels in heterokaryons. The lack of human beta-galactosidase expression in man-mouse somatic cell hybrids formed from fibroblasts of the infantile onset type disorder suggests that the defect is not corrected by the mouse genome. The ML(gal-neur-) disorder therefore appears to be a distinct subtype of the inherited neuraminidase deficiencies in which the defect mat occur in a post-translational or regulatory step which coordinately affects the expression of lysosomal beta-galactosidase and alpha-neuraminidase.

Defining the locus of origin of a genetically determined electrophoretic variant of a multilocus enzyme system; the Calcutta-1 of human LDH system is a B-locus variant.

Six (four Hindus, one Sikh, and one Muslim) outr of 213 individuals originating from different parts of the Indian subcontinent (namely, Andhra Pradesh, Maharashtra, Uttar Pradesh, East Punjab, and West Punjab) were found to be Calcutta-1 (CAL1) variants of lactate dehydrogenase (LDH). The CAL1 variant was originally described (and thus, generally believed at present) as an allelic variant at the LDHA locus in chromosome 11. By using an improved Cellogel electrophoretic procedure the isozyme patterns observed in the erythrocytes and leukocytes of the variant have indicated that the CAL1 is not variant of LDHA but that of LDHB, a chromosome 12 marker. The suggestion was supported by the isozyme patterns of LDH in a set of segregating clones of man-mouse somatic cell hybrids with the variant as human partner. Moreover, the variant cosegregated consistently with the human chromosome 12 and with the markers firmly assigned to the latter but not with human chromosome 11 or its markers in these hybrids. These results confirmed that the CAL1 is an LDHB variant.

Assignment of a structural gene for a fourth human diaphorase (DIA4) to chromosome 16 in man-mouse somatic cell hybrids

A diaphorase (DIA4), different from similar enzymes so far described in man, has been detected electrophoretically in human tissues and fibroblasts. The enzyme which is active both with NADH and NADPH was missing in erythrocytes. It was consistently undetectable in part of the diploid fibroblast cultures analyzed. The activity could be separated by Cellogel electrophoresis from rodent diaphorases. In manmouse somatic cell hybrids human DIA4 segregated with chromosome 16. This result indicates that its structural gene is located on this autosome. The enzyme exhibits similarities with a NAD(P)H dehydrogenase (EC 1.6.99.2) described in rat liver.

Localisation of the G gamma-, A gamma-, delta- and beta-globin genes on the short arm of human chromosome 11.

Human-mouse somatic cell hybrids have proved invaluable in assigning human genes to their respective human chromosomes. To date, the success of this approach has depended on identifying human proteins which are synthesised in hybrid cells containing a small number of human chromosomes. Consequently, chromosome assignment has been limited mainly to human proteins which are expressed in man-mouse somatic cell hybrids and for which a suitable assay, usually electrophoretic or immunological, exists to distinguish between the human and murine homologous proteins. This technique is therefore unsuitable for the assignment of those human genes which are expressed only in differential cells and not in hybrid cells. Here, we describe how nucleic acid hybridisation and restriction endonuclease mapping of DNA can be combined to test for the presence of human structural gene sequences within hybrid cell DNA. This method can be used to assign any purified human DNA sequence to a human chromosome, and does not require the DNA sequence to be expressed in man-mouse hybrid cells.

Gene assignment of alpha-fucosidase and glucose dehydrogenase to the p21 leads to pter region of chromosome 1 in man.

Assignment of human genes coding for alpha-fucosidase (alphaFUC) and glucose dehydrogenase (GDH) to chromosome 1 has been confirmed and a location in the p21 leads to pter region demonstrated using man-mouse somatic cell hybrids. The regional location of alphaFUC and GDH was established in cell hybrids using human cells possessing 1/2 translocation chromosomes [46,XX,t(1;2)(p21;q37)]. Hybrids which retained the 2q+ chromosome carrying the 1p21 leads to 1pter region concordantly expressed alphaFUC, GDH, and the short-arm markers ENO1, AK2, and PGM1. Hybrids which retained the 1p21 leads to 1qter region only expressed human PEPC and FH. Data obtained from hybrids in which spontaneous breaks in chromosome 1 had occurred indicate that the gene order in 1p21 leads to 1pter is (ENO1,GDH)-alphaFUC-AK2-PGM1.

Studies on the synthesis of immunoglobulins by man-mouse somatic cell hybrids

Studies on the synthesis of immunoglobulins by man-mouse somatic cell hybrids

Localization of a gene for human α-galactosidase B (=N-Acetyl-α-D-Galactosaminidase) on chromosome 22

The localization of the structural gene for human alpha-galactosidase B (= N-acetyl-alpha-galactosaminidase) was investigated by means of man-Chinese hamster and man-mouse somatic cell hybrids. The hybrid clones were analyzed for chromosomes and for a large number of known enzyme markers. The lysates of the hybrid cells were treated with Sepharose-coupled antihuman alpha-galactosidase B and the activity of the adsorbed enzyme was measured on the Sepharose beads as N-acetyl-alpha-galactosaminidase. The results show that the structural gene for human alpha-galactosidase B is situated on chromosome 22, and that there is no structural relationship between human alpha-galactosidase A and human alpha-galactosidase B.

Assignment of peptidase S (PEPS) to chromosome 4 in man using somatic cell hybrids.

A starch gel electrophoretic procedure is described that resolves peptidase S (PEPS) as well as the peptidases A, B, and C in man-rodent, rodent-rodent, and primate-rodent interspecific somatic cell hybrids. The interspecific PEPS cell hybrid phenotype can be resolved into a pattern which suggests that PEPS is composed of five or six identical subunits. Results are presented supporting assignment of the PEPS locus to chromosome 4 in man using man-mouse and man-Chinese hamster somatic cell hybrids. Human genes coding for peptidases A, B, C, and D were assigned to chromosome 18, 12, 1, and 19, respectively, confirming previous assignments. These somatic cell genetic data demonstrate the independent genetic control of the several human peptidases.

Correction of human mucolipidosis II enzyme abnormalities in somatic cell hybrids.

THE mouse genome has been used to correct the abnormal enzyme phenotype of a human neurodegenerative disorder and to provide information on the nature of the molecular defect. This has been demonstrated in man–mouse somatic cell hybrids using human cells derived from an individual with mucolipidosis II. Mucolipidosis II (ML II) or I-cell disease is a rare fatal childhood storage disease which is associated with deficiencies and electrophoretic abnormalities of several lysosomal hydrolases1–4. Somatic cell hybridisation provides a methodology for examining the expression of mutant genes and for identifying and mapping genes associated with human enzyme abnormalities. We have examined the isozyme patterns of six enzymes altered in this disorder in man–mouse hybrids formed with fibroblasts from an ML II patient. These altered enzymes showed corrected patterns of expression in cell hybrids. Since several human enzymes are corrected, a defect in the post-translational processing of lysosomal enzymes is suggested. Previous studies fusing human lysosomal storage disease cells with rodent cell lines have failed to demonstrate correction of the enzyme deficiencies by the rodent genome5–8. We present here the first example of the correction of an abnormal enzyme phenotype associated with a human lysosomal storage disease by fusing human and mouse genomes.

Intrachromosomal gene mapping in man: the gene for tryptophyl-tRNA synthetase maps in region q21 leads to qter of chromosome 14.

A gene for tryptophanyl-tRNA synthetase (EC 6.1.1.2), the enzyme which attaches tryptophan to its tRNA, has previously been assigned to human chromosome 14 by analysis of man-mouse somatic cell hybrids. We report here a method for the electrophoretic separation of Chinese hamster and human tryptophanyl-tRNA synthetases and its application to a series of independently derived Chinese hamster-human hybrids in which part of the human chromosome 14 has been translocated to the human X chromosome. When this derivative der (X),t(X;14) (Xqter leads to Xp22::14q21 leads to 14qter) chromosome carrying the human gene for hypoxanthine-guanine phosphoribosyltransferase was selected for and against in cell hybrid lines by the appropriate selective conditions, the human tryptophanyl-tRNA synthetase activity was found to segregate concordantly. These results provide additional confirmation for the assignment of the tryptophanyl-tRNA synthetase gene to human chromosome 14 and define its intrachromosomal location in the region 14q21 leads to 14qter. Our findings indicate that the genes for tryptophanyl-tRNA synthetase and for ribosomal RNA are not closely linked on chromosome 14.

Direct assignment of citrate synthase (CS) gene to human chromosome 12 in man-mouse somatic cell hybrids.

A panel of twenty independently derived clones of man-mouse somatic cell hybrids isolated from fusions involving eight different parent cell combinations simultaneously analyzed for human chromosomes, citrate synthase, and a large number of other enzyme markers firmly or tentatively assigned to individual human chromosomes have provided direct evidence for a firm assignment of the structural gene coding for citrate synthase (CS) to human chromosome 12.

Mapping <i>AK<sub>1</sub> ACO</i><i>N</i><sub>s</sub>, and <i>AK</i><sub>3</sub> to chromosome 9 in man employing an X/9 translocation and somatic cell hybrids

The adenylate kinase 1 (AK1), adenylate kinase3 (AK3), and aconitaseS (ACONS) genes have been assigned to chromosome 9 in man by employing an X/9 translocation segregating in man-mouse somatic cell hybrids. Segregation was controlled by taking advantage of the HAT/8-azaguanine selection-counterselection strategy directed at the X-linked HPRT locus. Assignment of AK1 to chromosome 9 has suggested the assignment of the ABO blood-group locus and the nail-patella (Np) locus to 9, since both loci are linked to AK1 by family studies.

The human PGM-2 and its chromosomal localization in man-mouse hybrids.

A detailed procedure is described for a rapid detection of phosphoglucomutase-2(=phosphopentomutase;PGM-2) on Cellogel following electrophoresis of extracts of human red blood cells and other tissues, including cultured fibroblasts and various types of primate-rodent somatic hybrid cells. The present study indicated that there is only one locus for phosphopentomutase in man. The data from a selected panel of 20 independent clones of man-mouse somatic cell hybrids, investigated for the presence of human chromosomes and for the presence or absence of human PGM-2 favored the assignment of the human PGM-2 locus to chromosome 4.

Simultaneous identification of chromatid replication and of human chromosomes in metaphases of man-mouse somatic cell hybrids. (With 1 color plate).

Simultaneous identification of chromatid replication and of human chromosomes in metaphases of man-mouse somatic cell hybrids. (With 1 color plate).