The Drosophila melanogaster Gart locus, known from previous work to encode the enzyme activity phosphoribosylglycinamide formyltransferase (GART), specifies two alternatively processed mRNAs and two proteins. We introduced the entire Gart locus into a Drosophila tissue culture cell line in which the locus is active. The resulting cell clones contained numerous copies of the locus and overproduced both mRNAs and both expected proteins, thus markedly facilitating analysis of these molecules. We assayed extracts of the clones for the activities of 10 enzymes important for de novo purine synthesis and found that, in addition to GART, two other purine pathway activities, phosphoribosylamine-glycine ligase (phosphoribosylglycinamide synthetase, GARS) and phosphoribosylformylglycinamidine cyclo-ligase (phosphoribosylaminoimidazole synthetase, AIRS), are similarly overproduced. AU three activities are present together on the larger overproduced protein. A smaller protein appears to possess only GARS activity. Therefore, alternative mRNA processing can allow cells to produce enzyme activities in forms that are either linked or unlinked to other activities.

No abstract

We describe the isolation and characterization of a Chinese hamster ovary cell line selected for resistance to N-carbamoyloxyurea. Using the mammalian cell permeabilization assay developed in our laboratory, a detailed analysis of the target enzyme, ribonucleotide reductase (EC 1.17.4.1), was carried out. Both drug-resistant and parental wild-type cells required the same optimum conditions for enzyme activity. The Ki values for N-carbamoyloxyurea inhibition of CDP reduction were 2.0 mM for NCR-30A cells and 2.3 mM for wild-type cells, while the Ki value for ADP reduction was 2.3 mM for both cell lines. Although the Ki values remained essentially unchanged, the Vmax values for NCR-30A cells were 1.01 nmoles dCDP formed/5 X 10(6) cells/hour and 1.83 nmoles dADP/5 X 10(6) cells/hour, while those for the wild-type cells were 0.49 nmoles dCDP produced/5 X 10(6) cells/hour and 1.00 nmoles dADP/5 X 10(6) cells/hour. This approximate twofold increase in reductase activity as least partially accounts for a 2.6-fold increase in D10 value for cellular resistance to N-carbamoyloxyurea exhibited by NCR-30A cells. The NCR-30A cell line was also cross-resistant to the antitumor agents, hydroxyurea and guanazole. No differences in Ki values for inhibition of CDP and ADP reduction by these two drugs were detected and cellular resistance could be entirely accounted for by the elevation in activity of the reductase in the NCR-30A cell line. The properties of N-carbamoyloxyurea-resistance cells indicate they should be useful for further investigations into the regulation of mammalian enzyme activity.

No abstract

The beta-casein level in untreated human milk is significantly higher than previously reported. The phosphoform distribution of beta-casein in individual donors varies widely. Anti-H. influenzae activity was detected in vitro among human beta-casein molecules with three or more phosphate groups.