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.

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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...