Autonomous replication in Drosophila melanogaster tissue culture cells

This study addresses the ability of DNA fragments from various sources to mediate autonomous DNA replication in cultured Drosophila melanogaster cells. We created a series of plasmids containing genomic DNA fragments from the Ultrabithorax gene of Drosophila and tested them for autonomous replication after transfection into Schneider line 2 cells. We found that all plasmids containing Drosophila DNA fragments were able to replicate autonomously, as were plasmids containing random human and Escherichia coli genomic DNA fragments. Most of the plasmids were detectable 18 days after transfection in the absence of selection, suggesting that transfected DNA is maintained in Drosophila cells without rapid loss or degradation. The finding that all plasmids containing Drosophila, human, or bacterial DNA replicate autonomously in Drosophila cells suggests that the signals that direct autonomous replication in Drosophila contain a low degree of sequence specificity. A two-dimensional gel analysis of initiation on one of the plasmids was consistent with many dispersed initiation sites. Low sequence specificity and dispersed initiation sites also characterize autonomous replication in human cells and Senopus eggs and may be general properties of autonomous replication in animal cells.     

The effect of a bacterial extract on synovial cells in tissue culture.

The studies reported here were undertaken to determine if Erysipelothrix crude extract (CE) could elicit consistent, detectable alterations in synovial cells in vitro. Synovial cells of rabbits were cultured in vitro and data about normal synovial cells in vitro were presented. Such cells were consistently and characteristically affected by exposure to small amounts of CE; an effect that was neutralized in the presence of specific and anti-CE anti-serum.With an increase in the amount of CE added to the cells in culture, a decreasing number of cells was formed but protein content remained relatively constant.     

Adrenal cells in tissue culture the effects of choleragen and ACTH on steroid and cyclic-AMP metabolism.

Primary cultures of mouse adrenocortical tumors provide a sensitive system for investigating the effects of the enterotoxin of the V. cholerae (choleragen) on cyclic-AMP metabolism in the intact cell. Like ACTH, the toxin stimulates the synthesis and release of steroids from these cells but its mode of action differs from that of ACTH. The steroidogenic response to ACTH is immediate and of limited duration. The initial rate of steroidogenesis is the highest. In contrast, the steroidogenic response to choleragen is preceded by a 30-240 minute lag period which is inversely related to the concentration of the toxin. Whereas prolongation of the response to a single dose of ACTH requires hormone concentrations above those producing maximal initial steroidogenic activity, persistent steroidogenesis is induced at all levels of the toxin. Steroidogenic responses are detectable with 10 pg/ml of choleragen or less. The respective effects of ACTH and choleragen on cyclic-AMP synthesis and release into the medium parallel those on steroidogenesis. Intracellular cyclic-AMP levels in ACTH-treated cells reach a peak within 20-30 minutes and decline to normal levels within 2-4 hours. In choleragen-treated cells, after the lage period, the levels of intracellular cyclic-AMP remain above control levels indefinitely. The effects of ACTH and choleragen on cyclic-AMP biosynthesis are additive at all levels of the two compounds. The effects of choleragen are blocked by prior treatment of the toxin with a five-fold molar excess of ganglioside GM1, a presumed constituent of the toxin-binding site.     

The effect of a bacterial extract on synovial cells in tissue culture

Summary The studies reported here were undertaken to determine if Erysipelothrix crude extract (CE) could elicit consistent, detectable alterations in synovial cells in vitro. Synovial cells of rabbits were cultured in vitro and data about normal synovial cells in vitro were presented. Such cells were consistently and characteristically affected by exposure to small amounts of CE; an effect that was neutralized in the presence of specific anti-CE antiserum. With an increase in the amount of CE added to the cells in culture, a decreasing number of cells was formed but protein content remained relatively constant. This work was supported, in part, by an NIH predoctoral fellowship, GM 42668, to P. H.     

Adrenal cells in tissue culture. VIII. Dissociation of the steroidogenic and glycolytic effects of cyclic nucleotides and adrenocorticotropin

Summary In monolayer cultures of mouse adrenal cortex tumor cells, high concentrations of 3′,5′-cyclic adenosine monophosphate and 3′,5′-cyclic cytidine monophosphate (1.0 to 10.0mm produce steroidogenic responses equivalent to maximally stimulating levels of adrenocorticotropin. 3′,5′-Cyclic guanosine monophosphate and other cyclic nucleotides are not steroidogenic. Although the steroidogenic action of adrenocorticotropin is accompanied by an increased rate of glycolytic activity, the cyclic nucleotides stimulate steroidogenesis without increasing glycolysis. The data suggest that adrenocorticotropin can effect certain alterations in adrenal metabolism by a mechanism which does not involve the adenyl cyclase system. Supported by grants from the American Cancer Society (P-395) and the National Institutes of Health (R01-AM09901). Presented in part at the 1969 Laurentian Hormone Conference, Mt. Tremblant, Quebec, Canada, August 28, 1969.     

The effect of tissue culture agar on chromosome breakage, sister-chromatid exchanges and clonogenicity in human cells

To investigate the cytogenetic effects of electromagnetic fields, a system containing an agar gel was developed to support the growth of various human cell types (peripheral lymphocytes, lymphoblasts, and fibroblasts). When compared to alioquots of identical cells, grown in plastic culture vessels, statistically significant increases in the frequencies of chromosome breakage, sister-chromatid exchange and decreased cloning efficiency were observed in those cells cultured in the agar. These results suggest a possible clastogenic and/or cytotoxic component in the agar gel.     

Effect of cyclophosphamide in vitro and on vaccinia virus replication in tissue culture.

The effect of cyclophosphamide on the growth of Vero, BSC-1, and HeLa cells in monolayer cultures was studied. By using hemocytometer counts and tritiated thymidine uptake as indicators of growth, it was found that cyclophosphamide significantly interfered with the metabolism of Vero and BSC-1 cells when sustained in Leibovitz medium. Vero cells and HeLa cells grown in Eagle medium were not affected by exposure to cyclophosphamide. Vaccinia virus replication in Vero cell monolayer cultures incubated with cyclophosphamide was markedly augmented, and this enhanced growth was reflected by virus quantitation techniques and metabolic studies using tritiated thymidine uptake. No difference in the distribution of infectious particles was found when cyclophosphamide-treated and control infected cultures were compared. Pathways other than through hepatic enzymes appear available to activate cyclophosphamide in vitro. These effects are dependent on both the cell type and the medium in which the cells are grown. Cyclophosphamide can facilitate vaccinia virus replication in vitro through metabolic interactions at the cellular level. The precise mechanisms underlying this effect require further study.