A Mammalian Cell Mutant with a Temperature-Sensitive Leucyl-Transfer RNA Synthetase

Abstract

A cell mutant of the Chinese hamster ovary line, which is temperature sensitive for protein synthesis, is specifically defective in vivo in its ability to charge tRNA with leucine. Cytoplasmic extracts exhibited temperature-sensitive leucyl-tRNA synthetase activity. It is, therefore, highly likely that the mutant has a structural alteration in leucyl-tRNA synthetase. The lowleakiness and low reversion rate of this mutant, combined with the specificity of the defect in its protein-synthesizing machinery, make it an appealing tool for investigating regulatory mechanisms in animal cells.Our understanding of biological processes at the molecular level has been advanced immeasurably by the judicious combination of genetics with biochemistry. This approach has played a far more important role in microbial systems; systems involving somatic mammalian cells have lagged behind, due in part to the paucity of well-defined mutations. We (2, 4) and others (3) have been developing selection procedures for the isolation of temperature-sensitive (ts) conditional lethal mutant lines of mammalian cells, and various mutant phenotypes isolated from various cell lines are now becoming available for study. Although there is indirect evidence that many ts as well as drug-resistant and nutritional-auxotrophic phenotypes are likely due to true mutations [see review by Thompson and Baker (4)], there have been only a few explicit demonstrations that altered gene products are associated with altered phenotypes (5). It is important to know whether ts mutant lines actually contain proteins (or other gene products) that are temperature sensitive, especially since some workers have proposed that altered phenotypes induced in culture might be explained by nongenetic modifications (6).We describe here a mutant of Chinese hamster ovary cells which has a temperature-sensitive leucyl-tRNA synthetase, the enzyme that adds leucine to tRNALU. A preliminary observation that protein synthesis declined rapidly and profoundly at the nonpermissive temperature attracted us to this mutant, as the protein-synthesizing machinery has been implicated in the control of proliferation of animal cells (7). Elucidation of the molecular basis for the phenotype of this mutant together with its stable genetic character render it very attractive as a device for investigating various molecular regulatory phenomena in animal cells. MATERIALS AND METHODSCells and Culture Conditions. The wild-type cells were a clone of the Chinese hamster ovary line (CHO) (8), which has a stable karyotype consisting of 21 chromosomes with little cell-to-cell variation (9). Cells from frozen stocks of this line and of the mutant tsHl were maintained in exponential growth in suspension culture at 34°. The growth medium was a-minimal essential medium (10) supplemented with antibiotics and 10% fetal-bovine serum (Flow Laboratories). Temperature was monitored with matched precision thermometers and was carefully controlled with water baths with a precision of 40.1'. The thermometers were ...

Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases.

“…CHO cell line Asn-5 has a mutation in the asnS gene, which renders asparaginyl-tRNA synthetase thermolabile and the cells nonviable at 39°C (2). UCW 56 is derived from the CHO cell line tsH-1 and has a mutation in the leuS gene which results in leucyl-tRNA synthetase being thermolabile (27,32). This cell line is also nonviable at 39°C.…”

Mitochondrial Protein Synthesis in a Mammalian Cell-Line with a Temperature-Sensitive Leucyl-tRNA Synthetase

“…The wild-type Chinese hamster ovary cell line (CHO) and the temperature-sensitive mutant (tsH1) were maintained in suspension culture in a-minimal essential medium [16] supplemented with 1004 fetal calf serum as described by Thompson et al [15]. The growth temperature for both cell lines was usually 34 "C, although CHO cells were occasionally grown at 37 "C. Both CHO and tsHl cells were generous gifts of Dr C. P. Stanners (The Ontario Cancer Institute, University of Toronto, Toronto, Ont.…”

“…Here we report a study with a mutant of the Chinese hamster ovary cell line (designated tsH1) shown previously by Thompson et ul. [15] to contain a temperature-sensitive leucyl-tRNA synthetase. At a non-permissive temperature, cytosolic protein synthesis was specifically inhibited and mitochondrial translation products could be selectively labelled.…”

Isolation and characterization of interspecific heat-resistant hybrids between a temperature-sensitive Chinese hamster cell asparaginyl-tRNA synthetase mutant and normal human leukocytes: Assignment of humanasnS gene to chromosome 18

“…Thus far mutants have been isolated with alterations affecting the ribosome (6)(7)(8)(9), an elongation factor (10,11), and numerous aminoacyl-tRNA synthetases (12)(13)(14)(15)(16). The majority of the aminoacyl-tRNA synthetase mutants have been isolated from the Chinese hamster ovary (CHO) cell line.…”