When 32P-labeled human recombinant immune interferon y (Hu-[32P]IFN-y) is crosslinked to human cells with disuccinimidyl suberateo a complex with a molecular
The establishment of a new glioma cell line, DBTRG-05MG, in a modified RPMI 1640 medium is described. The cells were derived from an adult female with glioblastoma multiforme who had been treated with local brain irradiation and multidrug chemotherapy; the tumor showed substantial change in histologic appearance compared to the original biopsy 13 mo. previously. The line has been successfully cryopreserved and passaged up to 20 times. The karyotype of the cells demonstrated it as a hypotetraploid line; the DNA index of 1.9 confirmed the karyotype analyses. By immunocytochemical analysis, the cell line reacted with polyclonal antibodies to vimentin, S100, and neuron specific enolase, reflecting its primitive neuroectodermal character. Positive immunostaining for epidermal growth factor receptor correlated with the excess of chromosome 7 seen in the karyotype. The cell line reacted negatively to antibodies against platelet-derived growth factor and its receptor, neuronal cell adhesion molecule, and glial fibrillary acidic protein. By flow cytometry, the cells were major histocompatibility class I antigen positive and class I antigen negative. Growth kinetic studies demonstrated an approximate population doubling time of 34 to 41 h and a colony forming efficiency of 71.4%. Western blot analysis showed the presence of low levels of normal-sized retinoblastoma protein. When compared to the patient's lymphocyte DNA, no loss of heterozygosity of the p53 tumor suppressor gene was observed in the DBTRG-05MG cell line DNA.
Erythropoietin (EPO), a glycoprotein hormone, is the major physiological regulator of erythrocyte production in mammals. A cDNA clone containing the entire human EPO-coding region was used for Southern blot analysis of a series of human-Chinese hamster somatic cell hybrids containing different combinations of human chromosomes. Synteny analysis revealed 100% concordance between the EPO gene and human chromosome 7. Further localization to the region qll-q22 was accomplished by in situ hybridization of 3H-labeled human EPO cDNA to metaphase chromosomes prepared from both human lymphocytes and the cell hybrid 879-2a that contained human chromosomes 5, 7, 9, 12, and 21. In addition, restriction fragment length polymorphisms were detected at a frequency of approximately 20% in a Chinese population using restriction enzymes either HindIII or Hinfl. These polymorphisms were inherited in-a Mendelian fashion. Thus, the EPO marker is reasonably polymorphic and should be useful in linkage analysis with other genetic markers on chromosome 7, including the locus for cystic fibrosis.Erythropoietin (EPO), a glycoprotein hormone, is the primary physiological regulator of erythrocyte production in mammals. Its main function is to control the level of circulating erythrocyte mass by promoting erythroid differentiation and initiating hemoglobin synthesis. In humans, it is produced primarily in adult kidney and in fetal liver (1-3). Under normal physiological conditions, the hormone circulates in the plasma at about 0.01 nM. Patients with chronic renal failure are anemic as a result of impaired renal function leading to low level of EPO production. It has been shown in sheep and rats that replacement therapy with EPO alleviates the anemic state (4, 5). Thus, therapy including EPO appears to be a rational approach to the treatment of anemia associated with chronic renal failure. The cloning of this important gene is crucial for the large-scale production of this hormone using modern biotechnological approaches (6-8). To provide a genetic basis for investigating the possible relationship between EPO and erythropoiesis, we have localized the EPO gene in the human genome by using a cDNA clone containing the entire EPO coding region for Southern blot analysis of a series of somatic cell hybrids containing different combinations of human chromosomes. In situ hybridization to metaphase chromosomes has also been carried out for the regional assignment of this gene. Furthermore, restriction fragment length polymorphisms (RFLPs) have been found at a frequency of approximately 20% in a human population and are shown to be inherited according to Mendelian principles.
MATERIALS AND METHODSSomatic Cell Hybrids. The human-hamster cell hybrids in Table 1 were constructed in several independent cell fusion experiments and have been described (9-14). The hybrid 32-1A was derived from a fusion between human and mouse A9 cells. The human chromosomal content in the hybrids was characterized by isozyme and karyotype analyses using both trypsin b...
Phenylketonuria (PKU) is an autosomal recessive disorder of amino acid metabolism caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH; phenylalanine 4-monooxygenase, EC 1.14.16.1). A cDNA clone for human PAH has previously been used to assign the corresponding gene to human chromosome 12. To define the regional map position of the disease locus and the PAH gene on human chromosome 12, DNA was isolated from human-hamster somatic cell hybrids with various deletions of human chromosome 12 and was analyzed by Southern blot analysis using the human cDNA PAH clone as a hybridization probe. From these results, together with detailed biochemical and cytogenetic characterization of the hybrid cells, the region on chromosome 12 containing the human PAH gene has been defined as 12q14.3----qter. The PAH map position on chromosome 12 was further localized by in situ hybridization of 125I-labeled human PAH cDNA to chromosomes prepared from a human lymphoblastoid cell line. Results of these experiments demonstrated that the region on chromosome 12 containing the PAH gene and the PKU locus in man is 12q22----12q24.1. These results not only provide a regionalized map position for a major human disease locus but also can serve as a reference point for linkage analysis with other DNA markers on human chromosome 12.
Human chromosome 6 encodes both the interferon gamma receptor as well as the class I major histocompatibility complex antigens, HLA-A, -B, and -C. However, the presence of chromosome 6 in somatic cell hybrids is insufficient to confer sensitivity to human interferon gamma (Hu-IFN-gamma) as assayed by class I HLA induction; it is necessary for both human chromosomes 6 and 21 to reside in the hybrid to generate a response to Hu-IFN-gamma. Treatment of such a hamster-human hybrid, Q72-18, with Hu-IFN-gamma induces the class I HLA antigens. Q72-18 cells selected by fluorescence-activated cell sorting for the loss of class I HLA induction also lost human chromosome 21. Fusions of such cells to a hybrid that contains only human chromosome 21 reconstitutes HLA antigen induction by Hu-IFN-gamma. Furthermore, fusions of hybrids containing a translocated human chromosome 6q and the HLA-B7 gene to a line containing only human chromosome 21 or a translocated 21q also reconstitutes HLA-B7 mRNA and antigen induction by Hu-IFN-gamma. Thus the segregation of cells on the basis of a biological effect by fluorescence-activated cell sorting and reconstitution by hybrid fusion provides a strategy by which some biological pathways can be mapped at a chromosomal level.
The reversion behavior of pleiotropic carbohydrate mutants, previously designated as ctr, was studied. The mutants revert to complete restoration of the wild-type phenotype, as well as to a spectrum of partial wild-type phenotypes. Lac+ reversions were found in the lac region (11 min) and some Mal+ reversions occurred at malB (79 min), at a distance from the site of the ctr mutations (46 to 47 min). About onethird of Lac+ and Mal+ revertants were constitutive for uptake of their respective substrates, and one-third modified for inducibility. The remaining third were not distinguishable from wild type. Induction of a ctr mutation in a lac constitutive strain, either operator or repressor mutant, did not affect lactose metabolism. A polar-like ctr mutant, deficient in both enzyme I and heat-stable protein of the phosphoenolpyruvate-dependent phosphotransferase strain was also described. Partial revertants of ctr were still found to lack enzyme I.
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