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

Cirullo, R E; Arredondo-Vega, Francisco X.; Smith, Moyra; Wasmuth, John J.

doi:10.1007/bf01543178

Cited by 20 publications

(12 citation statements)

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“…Two of these hybrids were of particular interest in this work since one (HHW It is interesting to note that the structural gene for asparaginyl-tRNA synthetase has recently been mapped to human chromosome 18 (30). Thus, there is no linkage between the structural gene for the enzyme responsible for asparagine biosynthesis and the structural gene for the enzyme responsible for its utilization in protein biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

Assignment of structural gene for asparagine synthetase to human chromosome 7

Arfin

Cirullo

Arredondo-Vega

et al. 1983

Somat Cell Mol Genet

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Section: Discussionmentioning

confidence: 99%

Assignment of structural gene for asparagine synthetase to human chromosome 7

Arfin

Cirullo

Arredondo-Vega

et al. 1983

Somat Cell Mol Genet

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“…The isolation and characterization of interspecific cell hybrids between normal human VOL. 3,1983 894 CIRULLO, DANA, AND WASMUTH leukocytes and either Asn-5 or UCW 56 has been described previously (6,8). HHW 105 is a hybrid between human leukocytes and UCW 56 and was selected at 39°C to require the human gene encoding leucyl-tRNA synthetase to be retained and expressed (8).…”

Section: Methodsmentioning

confidence: 99%

“…HHW 119 is a hybrid between human leukocytes and Asn-5, which was also selected at 39°C, to require retention and expression of the human gene encoding asparaginyl-tRNA synthetase. This cell line has retained three human chromosomes, numbers 12, 18, and 19 (6). Based upon heat inactivation experiments in vitro, HHW 119 contains both the mutant CHO asparaginyl-tRNA synthetase, which is thermolabile in vitro, and the human asparaginyl-tRNA synthetase, which is very stable in vitro (6).…”

Section: Methodsmentioning

confidence: 99%

“…Using this procedure, we focused on transferring the human genes encoding leucyl-and asparaginyl-tRNA synthetase into the appropriate CHO cell mutants because the presence of human DNA can be monitored against the background of rodent DNA by cytological as well as nucleic acid hybridization procedures (10,14,15,22). We have previously isolated interspecific somatic cell hybrids between human leukocytes and CHO cell mutants with temperature-sensitive leucyl-or asparaginyl-tRNA synthetases, which contain and express the corresponding human aminoacyl tRNA-synthetase genes (6,8,9). The expression of the human aminoacyl-tRNA synthetase genes in the hybrids results in their ability to grow vigorously at 39°C, a temperature which is nonpermissive for the mutant CHO parents.…”

Section: Transfer Of Human Genes Into Cho Cell Mutants 893mentioning

confidence: 99%

“…The expression of the human aminoacyl-tRNA synthetase genes in the hybrids results in their ability to grow vigorously at 39°C, a temperature which is nonpermissive for the mutant CHO parents. In both types of hybrids, the product of the human aminoacyl-tRNA synthetase gene can easily be distinguished from the product of the corresponding mutant CHO gene, based upon large differences in the thermolability of the different enzymes in vitro (6,8). Since these hybrids contained very few human chromosomes to begin with, they represented a significant enrichment of the human genes of interest.…”

Section: Transfer Of Human Genes Into Cho Cell Mutants 893mentioning

confidence: 99%

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

Cirullo¹,

Dana²,

Wasmuth³

1983

Mol. Cell. Biol.

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We have developed a simple and efficient procedure for transferring specific human genes into mutant Chinese hamster ovary cell recipients that does not rely on using calcium phosphate-precipitated high-molecular-weight DNA. Interspecific cell hybrids between human leukocytes and temperature-sensitive Chinese hamster cell mutants with either a thermolabile leucyl-tRNA synthetase or a thermolabile asparaginyl-tRNA synthetase were used as the starting material in these experiments. These hybrids contain only one or a few human chromosomes and require expression of the appropriate human aminoacyl-tRNA synthetase gene to grow at 39°C. Hybrids were exposed to very high doses of -y-irradiation to extensively fragment the chromosomes and re-fused immediately to the original temperature-sensitive Chinese hamster mutant, and secondary hybrids were isolated at 39°C. Secondary hybrids, which had retained small fragments of the human genome containing the selected gene, were subjected to another round of irradiation, refusion, and selection at 39°C to reduce the amount of human DNA even further. Using this procedure, we have constructed Chinese hamster cell lines that express the human genes encoding either asparaginyl-or leucyl-tRNA synthetase, yet less than 0.1% of their DNA is derived from the human genome, as quantitated by a sensitive dot-blot nucleic acid hybridization procedure. Analysis of these cell lines with Southern blots confirmed the presence of a small number of restriction endonuclease fragments containing human DNA specifically. These cell lines represent a convenient and simple means to clone the human genomic sequences of interest.The genes encoding various components of the protein synthetic machinery in mammalian cells represent a very large family offunctionally related genes. To begin understanding how the expression of this family of genes is coordinated, we are using a combined biochemical and genetic approach to analyze the complex process of protein synthesis. The large number of different Chinese hamster cell mutants that have been isolated with alterations in various protein synthesis components, including ribosomal proteins and aminoacyl-tRNA synthetases, makes this large family of genes especially suitable for genetic analysis and genetic manipulation (1,3,5,13,28,33,34). In Chinese hamster ovary (CHO) cells, two of these genes, leuS and asnS, which encode leucyl-tRNA synthetase and asparaginyl-tRNA synthetase, respectively, are particularly amenable to detailed genetic studies. Conditionally lethal, temperature-sensitive mutants with alterations in either of these genes can be isolated at very high frequencies (1,29,31). In addition, there is a simple counterselective system, growth at an elevated temperature, to isolate revertants in which the temperaturesensitive phenotype of the mutants has been suppressed by second-site intergenic or intragenic mutations. Because of the availability of large numbers of mutants and revertants, the leuS and asnS genes are most amenable to detailed studies o...

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