No abstract

Four genes encoding proteins designated as W, X, Yl, and Y2 were found previously to be amplified at different levels in a Chinese hamster fibroblast mutant line selected for overproduction of adenylate deaminase. To gain information on the molecular mechanisms responsible, we studied the levels of amplification and the structures of these four genes in several lineages of mutant cells with comparable activities of adenylate deaminase, the selected enzyme. Only the W gene was amplified in all the lines. In one line, the X, Y1, and Y2 genes were coamplified, while in others either the Yl gene or the pair X and Y2 were coamplified. The results were consistent with linkage of all the genes-in a particular order-in an amplifiable sequence with variable endpoints. Novel joints with a nonrandom distribution were observed. We frequently detected rearranged copies of the W gene, but very few novel joints were present in the other three genes in the six highly amplified lines examined. Some of the novel joints in gene W were highly amplified; they were generated by reamplification of a rearrangement that appeared at an early selection step. In some lines, reamplification was accompanied by deletion or mass correction of preexisting units. We discuss mechanisms which might account for these observations.We have shown previously (4) that unstable mutants overproducing adenylate deaminase (AMPD) can be isolated from Chinese hamster fibroblast line GMA32 by supplementing the growth medium with adenine, azaserine, and the AMPD inhibitor coformycin. Stepwise increases in the concentration of coformycin allow the recovery of mutants which accumulate increasing amounts of the target enzyme. Proteins other than AMPD also accumulate during these selections, and their levels return to those of wild-type cells in revertants with wild-type levels of AMPD activity. Plasmid cDNA probes specific for genes encoding four such proteins-designated W, X, Y1, and Y2-were isolated and used to show that the four genes are amplified at different levels in the AMPD-overproducing mutants studied in the previous experiments (5). We report here an analysis of the abundance, structure, and expression of these four genes in a variety of clones with comparable levels of AMPD activity, derived independently through multiple steps of selection. We again observed that the four genes were not coamplified to the same extent within the same line. Moreover, the pattern of amplification differed from clone to clone. In some cases, striking differences between the amplification and transcription levels of a gene were detected; they were shown to result from genetic rearrangements. The rearrangements were not randomly distributed along the amplified units, as judged from their preferential location in one of these genes. We discuss mechanisms able to generate the coamplification pattern observed in these clones. MATERIALS AND METHODSCell lines and mutant selection. The GMA32 line of Chinese hamster fibroblasts, conditions for its growth, and the details of stepw...

Pertussis toxin (PT) was found to elicit an increased thymidine uptake in resting B lymphocytes purified from human peripheral blood. A significant mitogenic effect was detected for toxin concentrations greater than 100 ng/ml (1nM) and a plateau of stimulation was reached at 1000 ng/ml (10 nM). B cell blasts, activated by a first signal such as Staphylococcus aureus Cowan I or insolubilized anti-mu chain antibody, were also stimulated to DNA synthesis by PT in the same range of concentrations. At lower sub-mitogenic concentrations, the toxin potentiated the response to the low-molecular weight B cell growth factor (LMW-BCGF or 12-kDa BCGF), a progression factor for activated B cells. The "A" or catalytic subunit was devoid of any activity on B cells, suggesting the stimulatory effect of the toxin might be associated with the binding or "B" subunit, as it has been shown for T cells. This hypothesis was strengthened by the observation that, as in T cell, the whole toxin but not the "A" promoter, was able to induce calcium influx in these cells. In addition, the purified "B" oligomer alone was found to promote DNA synthesis in B cells. Finally, a fragment of the soluble cleaved form of the CD23 molecule (Fc epsilon RII) could be involved in the process of PT mitogenicity for B cells.

The effects of transforming growth factor-beta (TGF-beta) on the proliferative response of human B cells to the low molecular weight B cell growth factor (BCGF) have been investigated in this study. It was found that TGF-beta, at picomolar concentrations, strongly inhibited the BCGF-induced proliferation of anti-mu chain or Staphylococcus aureus Cowan I-activated human B cells and also of a BCGF-dependent cell line derived from a human lymphocytic nodular lymphoma. This inhibitory effect was detected in normal and serum-free culture conditions. The suppression was greatly reduced when TGF-beta was added to the culture one day after BCGF and could be reverted by removing TGF-beta from the culture medium. Since TGF-beta has been detected in supernatants from activated T cells, this factor may represent an important regulatory molecule in the feedback control of B cell activation.