Multienzyme complex for metabolic channeling in mammalian DNA replication.

In the DNA-synthesizing phase (S phase) of CHEF/18 Chinese hamster embryo fibroblast cells, six enzymes associated with DNA metabolism, including DNA polymerase (deoxynucleoside triphosphate:DNA deoxynucleotidyl-transferase, EC 2.7.7.

Quiescent cells but not cycling cells exhibit enhanced actin synthesis before they synthesize DNA.

Major proteins synthesized by Swiss 3T3 cells at different stages of the cell cycle have been analyzed using double isotope labeling and one-dimensional SDS-polyacrylamide slab gels. The synthesis of actin was previously shown to be markedly enhanced a few hours after quiescent cells initiated growth following addition of serum. In contrast, the synthesis of actin remained at a constant rate, similar to that in quiescent cells, relative to synthesis of other proteins during the entire cell cycle.

Synthesis of labile, serum-dependent protein in early G1 controls animal cell growth.

We present a model to account for several major observations on growth control of animal cells in culture. This model is tested by means of kinetic experiments which show that exponentially growing animal cells whose ability to synthesize total protein has been inhibited with cycloheximide (by up to 70%) grow at rates approximately proportional to their rates of protein synthesis. However, virtually the entire elongation of the cell cycle occurs in the part of the G(1) phase that depends on a high concentration of serum in the medium.

Loss of epidermal growth factor requirement and malignant transformation.

Serum provides growth factors that regulate and limit the growth of normal cells in tissue culture. Animal cells that are malignantly transformed usually exhibit diminished serum requirements for growth in culture. We have used a defined, serum-free medium to determine which of these growth factors becomes dispensable for the growth of transformed Syrian and Chinese hamster fibroblast cells.

Role of nuclear size in cell growth initiation.

Swiss 3T3 cells arrested in B0 (quiescent state) by reducing serum content of the medium all contain the same amount of DNA but vary in nuclear volume over approximately a twofold range. By use of flow microfluorimetry, scatterplots of nuclear volume versus DNA content were obtained in intervals after serum stimulation. The earliest cells to enter DNA synthesis were those with the largest nuclei, whereas cells with the smallest nuclei were among the latest.

Changes in the synthesis of actin and other cell proteins after stimulation of serum-arrested cells.

The synthesis of both cytoplasmic and nuclear proteins has been studied as quiescent, serum-deprived Swiss mouse 3T3 cells are stimulated to transit the cell cycle. In serum-arrested cells a 200,000 dalton cytoplasmic protein and a 51,000 dalton nuclear protein were found to be preferentially synthesized. In serum-stimulated cells the first major protein whose synthesis was seen to increase had a molecular mass of 42,000 daltons.

Degradation of Escherichia coli DNA: evidence for limitation in vivo by protein X, the recA gene product.

DNA is more extensively degraded after it is damaged in recA mutants of E. coli than in wild type cells. All data presented here are consistent with the recA gene product, protein X, being an inhibitor of nalidixic acid induced degradation of the bulk DNA (but not of newly replicated DNA).

Growth control of normal and transformed cells.

Both serum factors and protein synthesis are required for normal cell growth. Swiss 3T3 cells require the serum growth factors insulin and EGF (epidermal growth factor) during the initial part of the G1 period, until they pass a restriction point about 2 h before the initiation of DNA synthesis. Concentrations of cycloheximide that inhibit protein synthesis by as much as 70% dramatically lengthen the cell cycle before the restriction point, while the cell cycle after the restriction point remains nearly constant.

2-amino-isobutyric acid and 3-O-methyl-D-glucose transport in 3T3, SV 40-transformed 3T3 and revertant cell lines.

In order to further investigate the connection between transport and growth control, 3T3 cells, SV40 transformed 3T3 cells (SV101), and three revertant cell lines derived from SV101 which have regained certain manifestations of growth control were used. Transport rates of 2-amino-isobutyric acid and 3-O-methyl-D-glucose were measured in sparse, confluent, serum-starved, and serum-stimulated cultures. As shown before, cessation of 3T3 cell growth in G0 under conditions of confluence or serum deprivation was associated with reduced rates of transport for both compounds, whereas the density and serum dependence of growth and transport was largely eliminated in SV101.

Inhibition of Escherichia coli division by protein X.

We propose that protein X provides the connection between damage to Escherichia coli DNA and inhibition of septation and cell division. This connection is needed to guarantee that each new bacterium receives a complete DNA copy. We present several new experiments here which demonstrate that the degree to which septation is inhibited following damage to DNA is correlated with the amount of protein X that is produced.

Control of DNA synthesis in tissue culture cells.

Eukaryotic DNA is functionally divided into thousands of replicons, each of which may be duplicated at a characteristic time within the DNA synthetic (S) period. Our approach toward an understanding of the molecular mechanisms which control orderly eukaryotic DNA synthesis has been: (a) to devise a method of cell synchrony in a suitable tissue culture system wherein all cells in the population enter and traverse the S period with a high degree of synchrony; (b) to determine, utilizing this system, precisely when during the S period critical events and macromolecular syntheses occur; and (c) to examine, by polyacrylamide-gel electrophoresis, the spectrum of proteins which become associated with chromatin during the S period in such a way as to suggest their involvement with DNA synthesis. Possible mechanisms for control are discussed based on the results presented here.

Animal cells reversibly permeable to small molecules.

A cell preparation, useful for studying the regulation of metabolism, was developed by making monolayer baby hamster kidney cells permeable. Hypertonically treated cells were permeable to nucleotides, but retained their gross cellular morphology, intact organelles, 100% of their DNA, and 91% of their total protein. The permeable cell synthesized DNA, RNA, and protein rapidly when supplied with the appropriate substrates and cofactors.