Abstract
We isolated interspecific somatic cell hybrids between human peripheral leukocytes and a temperature-sensitive CHO cell line with a thermolabile asparaginyl-tRNA synthetase. The hybrids were selected at 39 degrees C so as to require the expression of the human gene complementing the deficient CHO enzyme. In vitro heat-inactivation profiles of cell-free extracts from temperature-resistant hybrid cells indicate the presence of two forms of asparaginyl-tRNA synthetase. One form is very resistant to thermal inactivation, like the normal human enzyme, while the other form is very thermolabile, like the altered enzyme from the CHO parent. Hybrids and temperature-sensitive segregants derived from them were analyzed for the expression of known human chromosomal marker enzymes. The strong correlation between the expression of the human form of asparaginyl-tRNA synthetase and the presence of human chromosome 18 in hybrids suggests that the human gene, asnS, which corrects the heat-sensitive phenotype of the CHO asparaginyl-tRNA synthetase mutant, is located on chromosome 18.
Highlights
The process of protein synthesis in mammalian ceils is a complicated one, involving more than 100 proteins and over 50 unique RNA species
The temperature-sensitive Chinese hamster ovary (CHO) asparaginyl-tRNA synthetase (asnRS) mutant, Asn-5, was fused to human peripheral leukocytes as described in the Materials and Methods section and hybrids were selected based upon their ability to grow at 39~ This selection was, designed to isolate hybrid cell lines in which the defective
The interspecific temperature-resistant hybrid cell lines described in this report demonstrate that the temperature-sensitive phenotype of the CHO asparaginyl-tRNA synthetase mutant, Ash-5, can be corrected by a human gene, asnS
Summary
The process of protein synthesis in mammalian ceils is a complicated one, involving more than 100 proteins and over 50 unique RNA species. In order to better understand how the expression of this large group of functionally related genes is coordinated, more of the genes involved must be localized. Such a process will rely heavily on cell lines in which protein synthesis mutants can be selected. Cell lines derived from Chinese hamsters have been useful sources of protein synthesis mutants. The majority of the aminoacyl-tRNA synthetase mutants have been isolated from the Chinese hamster ovary (CHO) cell line. The phenotypes of all of these mutants are recessive in intraspecific cell hybrids and most have a temperature-sensitive, conditionally lethal phenotype which can be suppressed by high concentrations of the cognate amino acid [12, 17]
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