Erythromycin resistance in mouse L cells

The sensitivity of mouse cell lines in culture to the macrolide antibiotic, erythromycin stearate, was investigated. Both resistant and sensitive lines were found. Experiments indicated that in sensitive cells erythromycin stearate inhibits mitochondrial protein synthesis. Mutants resistant to erythromycin stearate were selected from the line LM(TK-), and these are also less sensitive to other macrolide antibiotics such as carbomycin and spiramycin. Attempts to transfer the erythromycin resistance of either the mutants or naturally resistant lines by fusion of cytoplasts with sensitive cells were unsuccessful, and it is concluded that resistance to erythromycin stearate is controlled by nuclear genetic factors.     

Carbomycin resistance in mouse L cells

A mutant has been isolated from the mouse cell line LM(TK-) which is stably resistant to the macrolide antibiotic, carbomycin. Mitochondrial protein synthesis in this mutant was carbomycin resistant and chloramphenicol sensitive. Fusions between carbomycin-resistant and -sensitive cells produced hybrids, most of which were sensitive to 10 microgram/ml carbomycin. At 7.5 microgram carbomycin/ml, the average population resistance is low initially but increases with time. Carbomycin-resistant cells were enucleated and fused with carbomycin-sensitive cells under a variety of selective regimes designed to allow growth of carbomycin-resistant cytoplasmic hybrids (cybrids). No transfer of carbomycin resistance via the cytoplasm was detected. Karyoplasts from carbomycin-resistant cells showed a low transfer of resistance to 7.5 microgram carbomycin/ml in karyoplast-cell fusions. Carbomycin resistance in this mutant is therefore most likely encoded in a nuclear gene.     

Introns are inconsequential to efficient formation of cellular thymidine kinase mRNA in mouse L cells.

TK mRNA levels were determined in mouse L cells transformed with intron deletion mutations of the chicken TK gene. Whether normalized per cell, per integrated gene, or per internal control signal, intron deletion did not diminish the efficiency of TK mRNA formation in transformed L cells. The results demonstrated that introns are not required for efficient biogenesis of cellular mRNA in transformed mouse L cells.     

Messenger RNA turnover in mouse L cells

The turnover of polyadenylic acid-containing messenger RNA and histone messenger RNA, which lacks poly(A), was studied in exponentially growing mouse L cells by measuring the kinetics of approach to steady-state uridine labeling. Constant specific activity of precursor pools was verified by showing that the data for stable RNA components, like ribosomal RNA and transfer RNA, follow theoretically predictable curves. In agreement with a previous report by Greenberg (1972), the data for poly(A)-containing mRNA (poly(A)(+)mRNA) follow theoretical curves for a class of molecules turning over with first-order (stochastic) kinetics. Cells growing with doubling times of 13·5 hours at 37 °C and 41 hours at 30 °C exhibited mean lifetimes for their poly(A)(+)mRNA of 15 hours and 42 hours, respectively, suggesting a parallelism between growth and turnover rates. The kinetic data for histone mRNA are not indicative of a stochastic process. Rather, they suggest an age-dependent decay or a zero-order (ordered) turnover with a mean lifetime of about six hours. One model, which gave a good fit to the data, considers that the histone messages persist for a fixed duration of the cell cycle, e.g. the DNA synthetic phase, and are then destroyed in a “sensitive period” after this phase. These results are discussed with regard to the possible implications of the poly(A) sequences in messenger RNA aging.     

Plasmodium falciparum: expression of the adenine phosphoribosyltransferase gene in mouse L cells

Genomic libraries of Plasmodium falciparum were constructed in the pBR322 plasmid. Using the DNA-mediated gene transfer technique, the genomic libraries were introduced into tissue-cultured mouse cells lacking the enzyme adenine phosphoribosyltransferase. Following selection for the adenine phosphoribosyltransferse phenotype, several colonies were isolated. All clones were shown to possess adenine phosphoribosyltransferase activity and pBR322 sequences. In addition, the Km value of adenine phosphoribosyltransferase (for adenine) from a transformant was found to be identical to that from P. falciparum. These results indicate that the adenine phosphoribosyltransferase gene of P. falciparum was successfully cloned and expressed in a mammalian system.     

Induction of DNA polymerase in mouse L cells

Two molecular species of DNA polymerase are found in mouse L cells. This study is concerned with the variation of these two species of enzyme with the rate of cell growth and DNA synthesis. The 3.4 S DNA polymerase, found in both nuclear and cytoplasmic fractions of mouse L cells, remains relatively constant during different stages of the growth curve. The heterogeneous 6 to 8 S DNA polymerase, found only in the cytoplasmic fractions, varies about 5 to 12-fold in correlation with DNA synthesis, as measured by [³H]thymidine incorporation.     

Expression of cotransferred genes in mouse L cells

The Herpes simplex virus (HSV) thymidine kinase (TK) gene was introduced into mouse L cells by cotransformation with the G418 resistance gene on plasmid pSV2neo.d5 and selection for antibiotic resistance. Fifty nanograms each of the TK gene and pSV2neo.d5 were mixed with 10 μg of carrier DNA and precipitated onto cells. Of 18 antibiotic-resistant colonies analyzed, 15 contained 1–5 copies of the TK gene and 13 of the 15 were phenotypically TK positive. These data demonstrate that it is possible to introduce small numbers of copies of cotransferred genes into cultured cells and that in most cases the genes are integrated in a manner compatible with expression.     

Distinction between malignant L cells and normal mouse fibroblasts by rosette formation with sheep red blood cells.

Murine L cell fibroblasts, and derivatives were found to rosette with sheep red blood cells (SRBC). Primary fibroblast explants from the parent murine strain, C3H, did not possess this potential. No rosettes were observed with primary fibroblast explants from C57BL and B10Br mice, with a human fetal lung fibroblast, with baby hamster fibroblasts or their polyoma transormed derivative, or with a cell line, 1T-22, derived from BALB/c mice. Hybridization of 1T-22 and L cells, by Sendai virus-mediated cell fusion, suppressed the rosette potential of the L cell parent. The receptor for SRBC on L cells appears to result from the expression of a recessive characteristic.     

DNA synthesis in permeabilized mouse L cells.

Mouse L cells are rendered permeable to nucleoside triphosphates by a cold shock with a near isotonic buffer. These cells retain their morphologic integrity and use exogenously supplied nucleotides and deoxynucleotides to synthesize RNA and DNA. The newly synthesized DNA is nuclear and is the product of semiconservative replication. Incorporation of deoxynucleotides into DNA by thymidine kinase-deficient cells were used to conform rigorously that the exogenously supplied deoxynucleotides were incorporated into DNA without intermediate processing through nucleosides. DNA synthesis requires the presence of Na+, ATP, all 4 deoxynucleotides, and Mg2+. The reaction is inhibited by N-ethylmaleimide, p-hydroxymercuribenzoate and actinomycin D. Hydroxy-urea and arabinosylcytosine do not inhibit the reaction whereas cytosine arabinoside triphosphate shows competitive inhibition with the deoxynucleotides. These findings indicate that the permeable cell system can be used for in situ evaluations of the replicative DNA polymerase using the endogenous DNA template.     

Persistence of mumps virus in mouse L929 cells

The characteristics of a persistent infection of L929 cells with mumps virus (MuV) is presented. The persistent infection (L-MuV cells) was regulated by interferon (IFN) produced endogenously and almost all the properties showed that the carrier culture was maintained by horizontal transmission of the virus. Small-plaque mutants, but not temperature-sensitive variants, were selected during the persistent infection. MuV released from L-MuV cells (MuV-pi) replicated efficiently in L929 cells, while infection of L929 cells with the original MuV-o resulted in an abortive infection. The efficient replication of MuV-pi in L929 cells can be explained by the findings that MuV-pi induced IFN more slowly and had lower susceptibility to IFN in L929 cells than MuV-o did. M protein was synthesized to a considerable degree in MuV-pi-infected cells, while it could not be detected in MuV-o-infected cells. By contrast, MuV-pi formed small plaques in Vero cell monolayers and the yield of MuV-pi in Vero cells was lower than that of MuV-o. M protein induced by MuV-pi decayed easily in Vero cells. M protein was considered to be a limiting factor for MuV replication in both cell lines.