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1.
The somatic cell hybrid method has been used to study the number and different types of human genes involved in the expression of adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) in normal cells and cells from a patient with ADA-deficient severe combined immunodeficiency disease (SCID). Genetic and biochemical characterization of ADA in SCID and the ADA tissue-specific isozymes in normal human cells indicates that additional genes, besides the ADA structural gene on chromosome 20, are involved in ADA expression. Human chromosome 6 encodes a gene, ADCP-1, whose presence is necessary for the expression of an ADA-complexing protein in human-mouse somatic cell hybrids [Koch, G. & Shows, T. B. (1978) Proc. Natl. Acad. Sci. USA 75, 3876-3880]. We report the identification of a second gene, ADCP-2, on human chromosome 2, that is also involved in the expression of the ADA-complexing protein. The data indicate that these two ADCP genes must be present in the same cell for that cell to express the complexing protein. Human-mouse somatic cell hybrids, in which the human parental cells were fibroblastss from an individual with ADA-deficient SCID, also required human chromosomes 2 and 6 to express the ADA-complexing protein, indicating that neither ADCP-1 nor ADCP-2 is involved in the ADA deficiency in SCID. The SCID-mouse hybrid cells expressed no human ADA even when human chromosome 20 had been retained. The deficiency of human ADA in these hybrids maps to human chromosome 20, and therefore is not due to the repression or inhibiton of ADA or its product by unlinked genes or gene products. We propose that the expression of the polymeric ADA tissue isozymes in human cells requires at least three genes: ADA on chromosome 20, ADCP-1 on chromosome 6, and ADCP-2 on chromosome 2. A genetic scheme is presented and the different genes involved in ADA expression and their possible functions are discussed.  相似文献   

2.
The purine and polyamine metabolic enzyme methylthioadenosine (MeSAdo) phosphorylase is abundant in normal cells and tissues but is lacking from many human and murine malignant cell lines and from cells of some human leukemias in vivo. To explore the genetic control of MeSAdo phosphorylase expression, we measured levels of the enzyme in somatic cell hybrids prepared by fusing MeSAdo phosphorylase-deficient mouse L cell lines with human fibroblasts. In the hybrid clones, MeSAdo phosphorylase activity segregated concordantly with adenylate kinase 1, a marker for human chromosome 9, but not with enzyme markers for any other human chromosome. In hybrid clones derived from human fibroblasts with a reciprocal translocation between chromosomes 9 and 17, MeSAdo phosphorylase activity was confined to cells containing the 9pter----9q12 region. In every case, the enzyme-positive hybrid clones displayed bands of MeSAdo phosphorylase activity with isoelectric points characteristic of both the human and murine enzymes. These results indicate that the structural gene for human MeSAdo phosphorylase, designated MTAP, can be assigned to the 9pter----9q12 region of human chromosome 9. Furthermore, these studies with interspecies somatic cell hybrids show that the MeSAdo phosphorylase-deficient state is recessive in mouse L cell lines.  相似文献   

3.
Hybrid cell clones between mouse cells deficient in thymidine kinase (EC 2.7.1.21) and two different human cell lines transformed by simian virus 40 (SV40) and deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) were examined for SV40 tumor (T) antigen(s). Concordant segregation of the gene(s) for SV40 T antigen and human chromosome C-7 was observed in these hybrids. The human chromosome C-7 which contains the gene(s) for SV40 T antigen is preferentially retained by the majority of the hybrid clones tested. When hybrid clones positive and negative for SV40 T antigen, derived from the fusion of SV40-transformed Lesch-Nyhan fibroblasts with mouse cells, were fused with CV-1 permissive cells, SV40-specific V antigen was observed only in the cultures derived from fusion of the hybrid clones positive for T antigen. This result indicates a linkage relationship between human chromosome C-7, SV40 T-antigen gene(s), and SV40 genome(s) integrated in the human transformed cells.  相似文献   

4.
In human tissues, adenosine deaminase (ADA) (adenosine aminohydrolase; EC 3.5.4.4) activity can be separated by gel electrophoresis into several isozymes. A structural gene (ADA) on chromosome 20 codes for the "erythrocyte" isozyme, ADA-1, which is also expressed in some nonerythroid tissues. Nonerythroid cells also differentially express five ADA "tissue isozymes" of a greater molecular weight than ADA-1. Each ADA tissue isozyme has a characteristic electrophoretic mobility and tissue distribution. It has been suggested that these ADA tissue isozymes are composed of ADA-1 and other components. We report that the expression of one of these tissue isozymes, ADA-d, is dependent upon ADA on chromosome 20 and another gene on chromosome 6 which functions in the assembly of the ADA tissue isozymes. In human-mouse hybrids segregating human chromosomes, chromosome 6(+),20(+) hybrids express both ADA-1 and ADA-d; chromosome 6(-),20(+) hybrids express only ADA-1; while 6(+),20(-) hybrids have no human ADA activity. ADA-d formation also occurs in vitro by self-assembly when an extract of human erythrocytes or chromosome 6(-),20(+) hybrids is mixed with a homogenate of chromosome 6(+),20(-) hybrids. The gene on chromosome 6, designated ADCP, codes for an adenosine deaminase complexing protein. The product of ADCP presumably combines with ADA-1 to form the ADA tissue isozymes. The data are consistent with the hypothesis that the distribution of enzymatic activity between ADA-1 and the tissue isozymes depends on the expression of the gene for ADA complexing protein, while the differences in the electrophoretic mobilities of the ADA isozymes, except ADA-1, are generated, as suggested by others, by the degree of glycosylation of the complexing protein.  相似文献   

5.
6.
Hybrid cell lines were obtained after fusion of mouse myeloid cells (WEHI-TG) with leukocytes from two patients with chronic myeloid leukemia. A third fusion was carried out with leukocytes from a patient with acute lymphocytic leukemia. All three patients carried the Philadelphia chromosome (Ph1) in the leukemia cell population. Cytochemical analysis confirmed the myelo-monocytic nature of the hybrid cell lines. The presence of Ph1 translocation products could be established in most hybrids derived from the two chronic myeloid leukemic patients, which confirms that indeed human myeloid cells were fused. Several of these hybrid lines showed reactivity with monoclonal antibodies known to be specific for human myeloid cells, whereas interlineage Chinese hamster fibroblast-human chronic myeloid leukemia hybrids failed to react with these antibodies. Five independently obtained monoclonal antibodies--MI/NI, UJ-308, VIM-D5, FMC-10, and B4.3--showed very similar reactivity patterns when tested on the hybrid clones. This result substantiates the evidence obtained from other studies, that these five antibodies are directed against the same myeloid-associated antigen. The gene(s) for expression of the latter antigen could be assigned to human chromosome 11.  相似文献   

7.
Human PRL (hPRL)-secreting adenoma cells obtained at hypophysectomy were fused with a mutant mouse fibroblast line (LMTK-) which is aminopterin sensitive due to a deficiency in the enzyme thymidine kinase. After fusion with polyethylene glycol, cells containing nuclear material from the two parental lines (heterokaryons) were selected in medium containing hypoxanthine, aminopterin, and thymidine, and resultant clones were screened for hPRL secretion. Functional human X mouse somatic cell hybrid clones secreting hPRL were isolated in order to study hPRL gene expression and regulation. Positive hybrid clones were subcultured and have sustained hPRL secretion. The hybrid nature of the cells was confirmed by fibroblastic morphology resembling the mouse parental cell, mixed karyotype of mouse and human chromosomes, and mixed isozyme banding pattern for human and mouse glucose-6-phosphate dehydrogenase and malic enzyme. Specific expression of the hPRL gene was demonstrated by the presence of electron microscopic secretory granules (650-800 nm), positive immunoperoxidase staining using anti-hPRL serum, and sustained secretion of immunoreactive hPRL, which comigrated with [125I] hPRL standard on Sephadex chromatography. Hormonal modulation of hPRL gene expression by TRH was dominantly expressed in the hybrid cell. Human chromosome 6 was identified in hybrid cells secreting hPRL, and the cells expressed human malic enzyme, a marker for this chromosome, thus confirming the chromosome assignment of the hPRL gene. The results show that functional replicating hybrids secreting hPRL can be isolated. The technique provides a useful in vitro model for the study of hPRL gene expression and modulation.  相似文献   

8.
Mutant human lymphoblast cells deficient in hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) activity were hybridized with thymidine kinase (EC 2.7.1.21)-deficient mouse fibroblasts. Hybrid cells were readily selected, as both parental lines were nonreverting and eliminated by hypoxanthine-amethopterinthymidine medium. Human lambda (lambda) chain was the only immunoglobulin chain produced by the lymphoblast parent, as determined by immunofluorescent techniques. Two independent hybrid clones chosen for detailed study synthesized human lambda chain, and continued to do so after prolonged culture.As in both parental lines, no human immunoglobulin heavy chains, complements C3 or C4, or alpha(1)-antitrypsin, or mouse immunoglobulin chains or complement C5 were detectable in the hybrids. Selection against thymidine kinase-containing hybrid cells with 5-bromodeoxyuridine did not eliminate positive lambda-chain reactivity, suggesting that the kinase and lambda-chain loci are not linked.The continued production of an immunoglobulin chain by human lymphoblast-mouse fibroblast hybrids contrasts with the extinction of other differentiated functions in several hybrid systems, and indicates that gene localization and linkage analysis for human immunoglobulin chains should be feasible with this system.  相似文献   

9.
A retroviral vector called SAX, containing the cloned human cDNA for adenosine deaminase (ADA), has been constructed and used to introduce the ADA gene into cultured T- and B-lymphocyte lines derived from patients with ADA deficiency. DNA analysis showed that the SAX vector was inserted intact into the T and B cells at approximately one copy per cell. The treated cells produced the characteristic isozymes of human ADA at a level similar to normal T and B lymphocytes. It is known that ADA-deficient lymphocytes are unusually sensitive to high levels of 2'-deoxyadenosine, and this is the mechanism thought to underlie the selective lymphocytotoxicity associated with ADA deficiency in vivo. Expression of the introduced ADA gene was sufficient to reverse the hypersensitivity of these genetically deficient lymphocytes to 2'-deoxyadenosine toxicity. These results support the suggestion that retroviral vector gene-delivery systems show promise for application to human gene therapy.  相似文献   

10.
The human lymphoblast line WI-L2 is subject to growth inhibition by a combination of the adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4.) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and adenosine. Although adenosine-induced pyrimidine starvation appears to contribute to this effect, uridine only partially reverses adenosine toxicity in WI-L2 and not at all in strain 107, an adenosine kinase-(ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) deficient derivative of WI-L2. Treatment of both cell lines with EHNA and adenosine leads to striking elevations in intracellular S-adenosyl-L-homocysteine (AdoHcy), a potent inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent methylation reactions. The methylation in vivo of both DNA and RNA is inhibited by concentrations of EHNA and adenosine that elevate intracellular AdoHcy. Addition of 100 muM L-homocysteine thiolactone to cells treated with EHNA and adenosine enhances adenosine toxicity and further elevates AdoHcy to levels approximately 60-fold higher than those obtained in the absence of this amino acid, presumably by combining with adenosine to form AdoHcy in a reaction catalyzed by S-adenosylhomocysteine hydrolase (EC 3.3.1.1). In the adenosine kinase-deficient strain 107, a combination of ADA inhibition and L-homocysteine thiolactone markedly increases intracellular AdoHcy and inhibits growth even in the absence of exogenous adenosine. These results demonstrate a form of toxicity from endogenously produced adenosine and support the view that AdoHcy, by inhibiting methylation, is a mediator of uridine-resistant adenosine toxicity in these human lymphoblast lines. Furthermore, they suggest that AdoHcy may play a role in the pathogenesis of the severe combined immunodeficiency disease found in most children with heritable ADA deficiency.  相似文献   

11.
Somatic cell hybrids between mouse peritioneal macrophages and HT-1080 human fibrosarcoma cells lose mouse chromosomes and retain the entire complement of human chromosomes. In contrast, somatic cell hybrids between cells derived from two different mouse continuous cell lines and HT-1080 human cells were found to lose human chromosomes preferentially. Loss of mouse chromosomes is not a general property of hybrids between mouse macrophages and transformed human cells; the hybridization of mouse macrophages with cells derived from five different human fibroblast lines transformed by simian virus 40 resulted in the production of hybrid clones that preferentially lost human chromosomes.  相似文献   

12.
Karyological examinations performed 24 hr to 11 days after Sendai virus-induced fusion of mouse cells of the permanent line 3T3 and human diploid cells, suggest that hybrids with extensive loss of human chromosomes derive from fusion involving all the chromosomes of both parental cells. Examination of 39 independent clones isolated from two different crosses between L mouse cells and cells of two permanent human lines, D98/AH2 and VA2, show that the karyotypes of human-mouse hybrids are a function of the interaction between the parental cells: the mouse chromosomal complement may be doubled or not. For the first time, viable hybrids that have retained almost all of the human chromosomes have been isolated.  相似文献   

13.
Somatic cell hybrids were generated by fusion of mouse erythroleukemia cells either to mouse L cells (B82), human fibroblasts (W1-18 VA2), or human marrow fractions enriched in erythroblasts. The hybrid cells were examined for globin gene expression by benzidine staining to detect cytoplasmic hemoglobin, and by molecular hybridization of cellular RNA to globin complementary DNA (cDNA) to detect globin messenger RNA (MRNA). The fibroblast (human or mouse) times erythroleukemia cell hybrids grown in monolayer retained most of the chromosomes of each parent. Neither cytoplasmic hemoglobin nor globin mRNA was detected in dimethylsulfoxide-treated fibroblast times erythroleukemia hybrid cells, indicating extinction of hemoglobin synthesis prior to the formation of cytoplasmic mRNA. The human marrow times mouse erythroleukemia hybrid cells grown in suspension culture contained only a few human chromosomes and exhibited low levels of hemoglobin synthesis which were amplified by 2% dimethylsulfoxide. Mouse (but not human) globin mRNA was demonstrated in these hybrid cells. The results suggest that somatic cell hybrids may be useful in searching for genetic factors which regulate activity of the globin genes.  相似文献   

14.
The inherited human disorders sialidosis and galactosialidosis are the result of deficiencies of glycoprotein-specific alpha-neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18; sialidase) activity. Two genes were determined to be necessary for expression of neuraminidase by using human-mouse somatic cell hybrids segregating human chromosomes. A panel of mouse RAG-human hybrid cells demonstrated a single-gene requirement for human neuraminidase and allowed assignment of this gene to the (pter----q23) region of chromosome 10. A second panel of mouse thymidine kinase (TK)-deficient LM/TK- -human hybrid cells demonstrated that human neuraminidase activity required both chromosomes 10 and 20 to be present. Analysis of human neuraminidase expression in interspecific hybrid cells or polykaryocytes formed from fusion of mouse RAG (hypoxanthine/guanine phosphoribosyltransferase deficient) or LM/TK- cell lines with human sialidosis or galactosialidosis fibroblasts indicated that the RAG cell line complemented the galactosialidosis defect, but the LM/TK- cell line did not. This eliminates the requirement for this gene in RAG-human hybrid cells and explains the different chromosome requirements of these two hybrid panels. Fusion of LM/TK- cell hybrids lacking chromosome 10 or 20 (phenotype 10+,20- and 10-,20+) and neuraminidase-deficient fibroblasts confirmed by complementation analysis that the sialidosis disorder results from a mutation on chromosome 10, presumably encoding the neuraminidase structural gene. Galactosialidosis is caused by a mutation in a second gene required for neuraminidase expression located on chromosome 20.  相似文献   

15.
We have studied somatic cell hybrids between thymidine kinase (EC 2.7.1.75) deficient mouse cells and human diploid fibroblasts for the expression of human acid alpha-glucosidase (EC 3.2.1.20). A deficiency in this enzyme is associated with the type II glycogenosis or Pompe disease. All 30 somatic cell hybrids selected in hypoxanthine/aminopterin/thymidine medium expressed human acid alpha-glucosidase and galactokinase (EC 2.7.1.6) and retained human chromosome 17; counterselection of the same hybrids in medium containing 5-bromodeoxyuridine resulted in the growth of hybrids that concordantly lost the expression of human acid alpha-glucosidase and galactokinase as well as human chromosome 17. Hybrids between thymidine kinase-deficient mouse cells and fibroblasts from a patient with Pompe disease that contained human chromosome 17 were found not to express human acid alpha-glucosidase. Because we have already shown that hybrids between mouse peritoneal macrophages and GM54VA simian virus 40-transformed human cells selectively retain human chromosome 17 and lose all other human chromosomes, we tested 13 independent mouse macrophage x GM54VA hybrid clones, including two that retained human chromosome 17 and no other human chromosomes, for the expression of human acid alpha-glucosidase and galactokinase. All 13 hybrid clones were found to express these human enzymes. Thus, we conclude that the gene coding for human acid alpha-glucosidase is located on human chromosome 17.  相似文献   

16.
Summary The metabolism of adenosine and its effects on phosphoribosylpyrophosphate, PP-ribose-P, dependent nucleotide synthesis were studied using erythrocytes from patients with adenosine deaminase and hypoxanthine phosphoribosyltransferase deficiency as models. The phosphorylation of adenosine was progressively inhibited by concentrations of adenosine greater than 1 µmol L–1 for control and ADA deficient erythrocytes. There was essentially no initial rate of phosphorylation at 30 µmol L–1 adenosine. Adenosine, 1 µmol L–1, also caused a 60% reduction in PP-ribose-P concentration in ADA deficient erythrocytes. For HPRT deficient erythrocytes in which ADA activity was blocked by coformycin, 10 µmol L–1 inosine stimulated PP-ribose-P dependent nucleotide synthesis from adenine, whereas, 10 µmol L–1 adenosine inhibited nucleotide synthesis. These observations suggest that adenosine phosphorylation and PP-ribose-P dependent nucleotide synthesis are inhibited under conditions in which adenosine accumulates, such as in hereditary or pharmacologically induced ADA deficiency.  相似文献   

17.
Prototrophic hybrids formed from an adenine-requiring Chinese hamster cell and human fibroblasts uniformly display new esterase activity that differs from that of either parental cell in electrophoretic mobility and substrate specificity. The hybrids that grew in the selective medium and possessed the new esterase activity had a single extra chromosome that resembled a B-group human chromosome. When clones of such hybrid cells were cultured in nonselective medium, they rapidly reverted to inability to synthesize adenine, disappearance of the new esterase activity, and simultaneous loss of the extra human chromosome. Esterase activity like that of the hybrid is present in cells of various Chinese hamster, but not human, tissues. It is postulated that particular Chinese hamster esterase genes became inactive after longterm cultivation, and that, in the hybrid cell, a human activator gene linked to the adeB gene and located on a human B-group chromosome reactivated expression of these Chinese hamster esterase genes.  相似文献   

18.
Human monoclonal antibodies (mAbs) were derived from lymph node lymphocytes and peripheral blood lymphocytes (PBL) from patients with melanoma. Four methods for generating human mAbs were compared: fusion with human [LICR-LON-HMy-2 (LICR-2)] or mouse (NS-1) cells; transformation by Epstein-Barr virus (EBV); and EBV transformation followed by NS-1 fusion. NS-1 fusion with lymph node lymphocytes resulted in a higher number of growing hybrids than LICR-2 fusion. Virtually no hybrids were obtained from NS-1 or LICR-2 fusions with PBL. EBV transformed lymphocytes from lymph node and peripheral blood with equal efficiency, and the yield of proliferating cultures for antibody screening was more than 10- to 30-fold greater than that obtained by fusion techniques. However, once antibody-producing cultures had been identified, stability and clonability of EBV-transformed cells were poorer than that of NS-1 hybrid cells. To combine the strengths of both methods, cultures of EBV-transformed cells were fused with NS-1; and hybrid clones were isolated that showed vigorous growth, clonability, and stable antibody secretion. Detailed specificity analysis of the mAbs produced by six of these clones indicated detection of a class 1 (unique) melanoma antigen, a class 3 melanoma antigen, and four ganglioside antigens (GD3, GM3, and two other, as yet uncharacterized, heterophile antigens).  相似文献   

19.
Cloning of cDNA sequences of human adenosine deaminase.   总被引:17,自引:5,他引:17       下载免费PDF全文
Cloned cDNA sequences of human adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) have been isolated from a cDNA library constructed in bacteriophage lambda gt10. The cDNA for the library was prepared from poly(A)+ RNA isolated from a human T-lymphoblast cell line, CCRF-CEM. The library was initially screened by differential plaque hybridization to labeled cDNA prepared from human T- and B-lymphoblast cell lines with a 21-fold difference in levels of translatable ADA mRNA. Two recombinants containing cloned cDNA sequences for ADA were identified by hybridization-selected translation. Both recombinants contained approximately 1,600 base pairs of inserted human DNA. Restriction maps of the two inserts were not identical. One contained approximately 40 base pairs of additional DNA toward the center of the cDNA. The cloned cDNA specifically hybridized to five fragments generated by HindIII digestion of human genomic DNA. It also hybridized to human lymphoblast RNA species 1.6 and 5.8 kilobases in length. The cDNA was used as a probe to estimate ADA mRNA levels in human lymphoblast cell lines. ADA mRNA levels correlate closely with levels of ADA catalytic activity and ADA protein in cell lines containing structurally normal ADA. A leukemic T-lymphoblast line produced 6 to 9 times as much ADA protein and ADA mRNA as transformed B-lymphoblast lines. Two mutant B-lymphoblast lines from patients with hereditary ADA deficiency contained unstable ADA protein but had 3 to 4 times the normal level of ADA mRNA.  相似文献   

20.
The human α-globin and phosphoglycollate phosphatase (EC 3.1.3.18) genes have been regionally localized to the short arm of human chromosome 16 (HC16). This was accomplished by fusing mouse fibroblasts (A9) to human fibroblasts that contain a reciprocal translocation between the long arms of chromosomes 16 and 11. The murine A9 cells are deficient in adenine phosphoribosyltransferase (APRT), an enzyme present on the long arm of HC16 (HC16q). Hybrid cells were grown in selection culture medium that required the cells to retain human APRT. Therefore, the hybrids exhibited stable retention of the entire HC16 or the rearranged chromosome containing HC16q. We isolated five independent primary and secondary hybrid cell lines which retained either HC16 or HC16q at a high frequency. The presence of human α-globin genes in the various clones was established directly by DNA extraction and hybridization to a cDNA probe for human α-globin genes. Autoradiographs showed that hybrid cells containing the long arm, but not the short arm, of HC16 showed only the background mouse bands. Hybrid cells that retained the entire HC16 demonstrated the band(s) containing the human α-globin genes. Hybrid cells that contained HC16 with its α-globin genes were then placed in culture medium that contained diaminopurine, which is lethal for cells containing APRT. These counter-selected hybrid cells had lost HC16 and also lost the human α-globin genes as determined by blot hybridization. The presence of α-globin gene sequences in the hybrid clones was concordant with HC16 only and not with any other human chromosome. These results confirm the assignment of α-globin genes to HC16 and localize the genes to the short arm. We also assign the locus for phosphoglycollate to the short arm of HC16.  相似文献   

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