We have previously transferred an ovalbumin-beta-globin fusion gene (ovalglobin) into primary cultures of chick oviduct cells and demonstrated that an ovalbumin gene 5'-flanking sequence between -221 and -95 is necessary for progesterone-mediated transcriptional induction (Dean, D. C., Knoll, B. J., Riser, M. E., and O'Malley, B. W. (1983) Nature (Lond.) 305, 551-554). Here we compare 5'-flanking sequences required for induction of the ovalglobin gene by 17 beta-estradiol and progesterone. The early gene of simian virus 40 was inserted into the same plasmid as the ovalbumin fusion gene to serve as an internal control. Since transcription of the viral early gene was unaffected by the presence of steroid hormone or deletions in the ovalbumin gene 5'-flanking region, the level of its transcripts could be monitored as a reference standard for ovalglobin transcription. Ovalglobin transcripts initiated principally from the ovalbumin cap site in the presence or absence of progesterone and 17 beta-estradiol. Deletion of 5'-flanking sequences to -197 had little effect on the induction with either hormone, while successive deletions to -180, -161, and -143 resulted in a gradual decrease in the level of induction. Deletion to -95 eliminated the induction. The results of this study indicate that DNA control elements for regulation of the ovalbumin gene by estrogen and progesterone either overlap directly or are clustered in close proximity in the 5'-flanking region near the ovalbumin gene promoter.

The gene for the alpha subunit of human chorionic gonadotropin (hCG) has been tentatively assigned to human chromosome 18. This localization was accomplished through the use of Southern blot analysis. A full-length cDNA probe for the hCG alpha subunit and DNA isolated from a series of somatic hybrids between mouse and human cells were utilized to make this assignment. In addition, in situ hybridization with normal human peripheral blood lymphocytes as a source of human chromosomes and with the same cDNA probe confirmed this result. The presence of human chromosome 18 was required for the detection of DNA fragments characteristic of the alpha-hCG gene. These results are consistent with our previous observation that human chromosomes 10 and 18 are required for the production of hCG in cultured cells.

The objective of this work was to isolate cultured mouse cells with amplified adenosine deaminase genes. Such cell lines should be very useful in an effort to obtain the protein and nucleic acid probes required to study adenosine deaminase gene structure and regulation. Since adenosine deaminase expression is not required for growth of cells in culture, the first step necessary to isolate adenosine deaminase gene amplification mutants was to devise selective conditions in which adenosine deaminase activity was required for survival. This was accomplished by developing a new selection system, termed 11AAU, which selected simultaneously for adenosine deaminase and adenosine kinase. The 11AAU selection medium consists of alanosine (0.05 mM) to block de novo AMP biosynthesis, adenosine (1.1 mM) to provide a salvage route for AMP biosynthesis via the adenosine kinase reaction, and uridine (1.0 mM) to alleviate the block in UMP biosynthesis caused by adenosine at the concentration employed. Because adenosine is highly cytotoxic at 1.1 mM, adenosine deaminase expression is required to detoxify excess adenosine by converting it to inosine. We used 11AAU selection in conjunction with stepwise selection for increasing resistance to deoxycoformycin, an adenosine deaminase inhibitor, to obtain highly drug-resistant cells with a 6000-fold increase in adenosine deaminase activity. Adenosine deaminase accounted for approximately 50% of the soluble protein in highly drug-resistant lines and was indistinguishable from that in the parent as judged by isoelectric focusing, electrophoretic mobility on starch gels, and by deoxycoformycin binding studies. Increased adenosine deaminase was also correlated with the presence of numerous double-minutes, cytogenetic structures indicating the presence of amplified DNA. Growth in the absence of selection was accompanied with the loss of double-minutes and a ten-fold decline in adenosine deaminase levels. Based on the stepwise selection protocol employed, the instability of the phenotype, and the presence of double-minutes, we believe that the increased adenosine deaminase is most likely the result of amplification of adenosine deaminase genes.

JEG-3 is a human choriocarcinoma cell line characterized by low levels of adenosine deaminase expression. For the purpose of studying adenosine deaminase gene regulation in the JEG-3 cells, we attempted to select variant cells having increased adenosine deaminase expression. This was accomplished by selecting cells for resistance to the cytotoxic adenosine analogs 9-beta-D-arabinofuranosyl adenine (ara-A) or 9-beta-D-xylofuranosyl adenine (xyl-A), both of which could presumably be detoxified by the action of adenosine deaminase. Single step high dose selection was ineffective in obtaining cells with increased adenosine deaminase. However, multistep selection using either ara-A or xyl-A resulted in cell populations with increased adenosine deaminase activity. Removal of selective pressure resulted in decreased adenosine deaminase levels. Subclones of xyl-A-resistant cells belonged to one of three phenotypic classes characterized by either elevated adenosine deaminase levels, decreased adenosine kinase levels, or both of these features. One subclone (A3-1A7) with unaltered adenosine kinase expression showed a 20-fold increase in adenosine deaminase expression. Further selection of this subclone for increasing xyl-A resistance resulted in an additional 2-fold increase in adenosine deaminase expression, followed by loss of adenosine kinase expression. These adenosine kinase-deficient cells showed no subsequent increase in adenosine deaminase expression in response to further xyl-A selection pressure. These results confirmed that xyl-A toxicity was mediated through its phosphorylated form and indicated that resistance may result from increased adenosine deaminase levels and/or adenosine kinase deficiency. The increased adenosine deaminase expression of the A3-1A7 subclone was exclusively in the ADA 2 allelic form. However, cell fusion experiments between A3-1A7 cells and mouse C1-1D cells established the existence of functional copies of both ADA 1 and ADA 2 allelic genes in the A3-1A7 cells. The increased expression of only one of the two functional ADA alleles, the requirement for a stepwise selection protocol to obtain cells with increased adenosine deaminase, and the instability of the adenosine deaminase phenotype in the absence of selective pressure suggest that the alteration of adenosine deaminase phenotype in the drug-resistant cells was the result of adenosine deaminase gene amplification.

We have established somatic cell hybrids by fusing cells from two human insulinomas with an established murine cell line LMTK- Cl1D. After selection of the hybrids, the media were analyzed and found to contain insulin and human C-peptide immunoreactive material. The newly synthesized material was further characterized by pulse labeling and immunoprecipitation, and shown in five hybrid lines on Sephadex G-50 chromatography to have a size similar to proinsulin. The hybrids produced the apparent proinsulin-like material for up to 7 mo. Chromosome composition of the hybrids was determined by isozyme analysis and banding techniques. Chromosome 11, which previously has been assigned the insulin gene using cDNA probes, was identified in the hybrids producing proinsulin-like material. However, the retention of this chromosome did not always assure the production of hormone. This independent technique has confirmed the localization of the insulin gene to chromosome 11 and offers the opportunity of studying insulin processing and developing continuous insulin-producing cell lines.