Replicating Units (Replicons) of DNA in Cultured Mammalian Cells

Exponentially growing L5178Y mouse leukemic cells were incubated in the presence of 5′-bromodeoxyuridine (BUdR) for about 4 hr, transferred to the nonBUdR-containing medium for a certain period (t hours), and then pulse-labeled with TdR-³H for 10 min. When DNA isolated from these cells was subjected to CsCl gradient centrifugation, the ³H-activity was found to shift gradually from the heavy BUdR-containing peak to the light nonBUdR-containing peak with increasing time t. The average time required for the complete shift of ³H-activity from the heavy to the light DNA fraction was 2.76 hr. Taking this as the average replicating time and the size of DNA fragments in the present preparation as 1.3 × 10⁷ daltons, the rate of replication was found to be 2.1 nucleotides per strand per replicon per sec. By taking the upper limit of the average replicating time as the S period (7.3 hr), various characteristics of the replicating units, such as the lower and upper limits of average size, the average replicating time, the average number of replicating units, etc., were calculated (see Table I).     

Radiation-Induced Breaks of DNA in Cultured Mammalian Cells

Mouse leukemic cells (L5178Y) in suspension culture were irradiated and the extent of single-strand breaks and double-strand cuts of DNA was estimated by sucrose gradient centrifugation. The radiation produced 3.0 single-strand breaks per cell (G(1) stage) per rad and approximately 0.3 double-strand breaks per cell (G(1) stage) per rad.     

Growing Points of DNA in Cultured Mammalian Cells

A fraction of DNA, known to be replicating, was examined in the electron microscope. Characteristic branch points were identified as sites at which replication had been taking place. No localized strand separations, nor other structural alterations were discernible at these points.     

Characterization of the Rapidly Labeled Hybridizable RNA Synthesized in L5178Y Mouse Leukemic Cells: Hybridization Lifetime Studies

An attempt is made to characterize the rapidly labeled hybridizable RNA of L5178Y mouse leukemic cells which has been shown to have similar base sequences when synthesized in two different stages of the cell cycle. The size of rapidly labeled RNA molecules was heterogeneous. For labeling times of 20 min or less, the per cent of hybridization was maximal. With longer labeling times, the per cent of hybridization decreased as radioactivity appeared in long-lived species of low hybridization efficiency; the radioactivity profile resembled the optical density profile in sucrose gradients. The lifetime of newly synthesized hybridizable RNA was studied by pulse labeling exponentially growing cells and then “chasing” with nonradioactive uridine. The per cent of hybridization was studied as a function of chase time. Three RNA groups, which comprised different proportions of rapidly labeled hybridizable RNA, were distinguished. The short-lived group had a half-life of 10 min, much less than the values reported in the literature for messenger RNA of mammalian cells. The half-life of 1-1½ hr observed for a medium-lived group more closely corresponds to that of messenger RNA. A long-lived group had a half-life of approximately 20 hr. Specific activity measurements during chase indicate the presence of a “pool” of labeled uridine derivatives. The uridine of this pool appears to be nonexchangeable with but dilutable by exogenous uridine. A nontoxic concentration of actinomycin D was added to the chase media in an attempt to block the “pool effect”. A rapidly degradable RNA was demonstrable both by specific activity and per cent of hybridization measurements.     

Specific G2 phase toxicity of chloramphenicol on mouse leukemic cells (L5178Y)

The effect of chloramphenicol on progression through the cell cycle of L5178Y cells was investigated. Using eosin staining as a viability index, G2 cells were shown to be specifically killed at a concentration of chloramphenicol generally used to study mitochondrial protein synthesis. Pretreating cells with chloramphenicol induced resistance to this G2 lethality.     

STATIONARY PHASE OF CULTURED MAMMALIAN CELLS (L5178Y)

The stationary phase of the mammalian cells L5178Y in culture can be divided into two stages: (a) an early phase characterized by the decline of mitotic index, followed by a stabilization of the cell number, and (b) a late stage, occurring several hours after the flattening of the growth curve, during which dead or dying cells appear in the cultures. The estimates of rates of cell progress showed that the rates from G₁ to S and from G₂ to M were affected in the early stationary phase. The main cause of cessation of increase in cell number in the early stationary phase is resulted from the decline in mitotic index, which is caused by prolongation of the G₂ period. The importance of the G₂ stage in regulating the cell growth is discussed in relation to other known situations in the literature.     

Amino acid content of L5178Y and L5178Y/L-ASE cells after L-asparaginase treatment

The amino acid contents of tumor cells that are either sensitive or resistant to treatment with L-asparaginase were measured. These amino acid concentrations were measured as a function of incubation time with L-asparaginase or as a function of the L-asparaginase dose. The cell types compared were the mouse leukemia lines L5178Y (sensitive to L-asparaginase treatment) and L5178Y/L-ASE (resistant to L-asparaginase treatment). Upon L-asparaginase treatment both cell lines lost most of their cellular asparagine but, whereas the resistant cells exhibited the ability to rebound to about 50% of initial values, the sensitive cells did not. While previous work had suggested that asparagine-dependent glycine synthesis was essential for sensitive cells (but not in resistant cells), we found no difference in the glycine content of either of the two cell lines as a function of either time or dose that would support this hypothesis. Major differences between the two cell lines were seen in the content of the essential amino acids before treatment with L-asparaginase. After incubation without L-asparaginase the contents of the two cell lines became similar. These results are discussed in terms of possible mechanisms of L-asparaginase sensitivity and resistance.     

Isolation of temperature-sensitive mutants from murine leukemic cells (L5178Y)

Two temperature-sensitive mutants (ts1 and ts3) have been isolated from murine leukemic cells, L5178Y, after mutagenesis and cytosine arabinoside selection. Both ts1 and ts3 grew normally at the permissive temperature (33 °C) but not at the non-permissive temperature (39 °C). Consistent results were obtained with the growth patterns in suspension culture as well as the plating efficiencies in soft agar. Temperature shift experiments showed that the mutant cells remained viable after extended exposure to the non-permissive temperature. Labeling studies with radioactive precursors indicated that the synthesis of DNA, but not of RNA or protein, was affected in these mutants at 39 °C. The defective function of ts3 cells was substantially corrected by supplementing alanine, hypoxanthine, and pyruvate.     

Proteins binding to DNA and their Relation to Growth in Cultured Mammalian Cells

Analysis of the proteins of mouse fibroblasts which can bind to DNA suggests that one of them may control DNA synthesis.     

Inhibition of Growth of L5178Y Leukemia Cells by a Fungal Folate-deaminating Enzyme

Reactive sites of adzuki bean proteinase inhibitor II were determined by limited hydrolyses with catalytic amounts of trypsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1] at pH 3.0. Treatment of the trypsin-modified inhibitor with carboxypeptidase B [EC 3.4.12.3] released lysine from the inhibitor and led to complete loss of the activity for trypsin, virtually, without affecting the chymotrypsin-inhibitory activity. Limited hydrolysis with chymotrypsin resulted in a selective cleavage of a single tyrosyi peptide bond in the inhibitor, and treatment of this modified inhibitor with carboxypeptidase A [EC 3.4.12.2] abolished the chymotrypsininhibitory activity, having no effect on the trypsin-inhibitory activity. After reduction and S-carboxymethylation, the trypsin- and the chymotrypsin-modified inhibitors both could be separated into two components by gel-filtration on Sephadex G–50 and DEAE-cellulose chromatography. Amino acid and end group analyses of these components indicated that the reactive sites of inhibitor II are the Lys²⁷-Ser²⁸ bond against trypsin and the Tyr⁵⁴-Ser⁵⁵ against chymotrypsin.

Chemical modification of inhibitor II with cyanogen bromide had a fatal effect on the inhibitory activity against trypsin but no effect against chymotrypsin.     

Novobiocin treatment reverses radiation-induced alterations in higher-order DNA structure in L5178Y nucleoids

We have studied the effect of novobiocin treatment on radiation-induced damage and its repair in higher-order DNA structure in two mouse leukemia cell lines differing in their radiosensitivity, L5178Y-R (LY-R) and L5178Y-S (LY-S). We used the fluorescent halo technique to measure alterations in the superhelical density and the topological constraints of DNA in LY-R and LY-S nucleoids. The results for untreated cells show that both cell lines reached maximal DNA unwinding at the same concentration of propidium iodide (PI), whereas LY-S nucleoids were less efficient in their ability to rewind their DNA. The loop size did not differ significantly between the cell lines. Incubation of LY-R and LY-S cells with novobiocin at a concentration which does not influence survival (0.1 mM for 45 min), but inhibits DNA synthesis in LY-R cells (by 28%) to a greater extent than in LY-S cells (by 10%), also causes more DNA unwinding in LY-R nucleoids than in LY-S nucleoids. However, a decreased superhelical density was observed in nucleoids from both cell lines. Novobiocin applied before, and present during, irradiation prevents radiation-induced alterations in DNA supercoiling more efficiently in LY-R than in LY-S cells. The presence of novobiocin during the repair period increased DNA rewinding to levels not significantly different from control values in nucleoids from both cell lines.     

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.