The dose-response relationship for ethyl methanesulfonate-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells

Hsie, Abraham W.; Brimer, Patricia A.; Mitchell, Thomas W.; Gosslee, D.G.

doi:10.1007/bf01538449

Cited by 187 publications

(45 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The mouse retinoblastoma cell line 661 was developed from the retinal tumor of a transgenic mouse expressing SV40 large T antigen under the control of the human interphotoreceptor retinoid-binding protein (IRBP) promoter (24). 661TG r was derived from 661 by ethyl methanesulfonate mutagenesis (400 g͞ml EMS for 18 h) and selection in 5 g͞ml 6-thioguanine (25). Lymphoblastoid cell lines were established by Epstein-Barr virus transformation of peripheral white blood cells (26).…”

Section: Methodsmentioning

confidence: 99%

3′ deletions cause aniridia by preventing PAX6 gene expression

Lauderdale

Wilensky

Oliver

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

145

116

View full text Add to dashboard Cite

Aniridia is a panocular human eye malformation caused by heterozygous null mutations within PAX6, a paired-box transcription factor, or cytogenetic deletions of chromosome 11p13 that encompass PAX6. Chromosomal rearrangements also have been described that disrupt 11p13 but spare the PAX6 transcription unit in two families with aniridia. These presumably cause a loss of gene expression, by removing positive cis regulatory elements or juxtaposing negative DNA sequences. We report two submicroscopic de novo deletions of 11p13 that cause aniridia but are located >11 kb from the 3 end of PAX6. The clinical manifestations are indistinguishable from cases with chain-terminating mutations in the coding region. Using human ؋ mouse retinoblastoma somatic cell hybrids, we show that PAX6 is transcribed only from the normal allele but not from the deleted chromosome 11 homolog. Our findings suggest that remote 3 regulatory elements are required for initiation of PAX6 expression.

show abstract

Section: Methodsmentioning

confidence: 99%

3′ deletions cause aniridia by preventing PAX6 gene expression

Lauderdale

Wilensky

Oliver

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

145

116

View full text Add to dashboard Cite

show abstract

“…The parental line, CHO-K1-BH4 (hgprt+), has been previously described (6). The X3/5 line (HGPRT-), which served as the transformation host for the pSV2gpt gene transfer experiments, was derived from the CHO-K1-BH4 line after an X-ray irradiation of 500 rads and selection in 10 ,uM 6-thioguanine; several lines of evidence indicate that it carries a stable, nonrevertible mutation at the hgprt locus (R. Machanoff, M.S.…”

mentioning

confidence: 99%

Detection of deletion mutations in pSV2gpt-transformed cells.

Tindall¹,

Stankowski²,

Machanoff³

et al. 1984

Mol. Cell. Biol.

128

View full text Add to dashboard Cite

We have developed a system to study mutations that affect xanthine-guanine phosphoribosyltransferase gene (gpt) expression in hypoxanthine-guanine phosphoribosyltransferase-deficient CHO cells that have been transformed by the plasmid vector pSV2gpt. One isolated transformant, designated AS52, carries a single copy of the Escherichia coli gpt gene stably integrated into the high-molecular-weight DNA and expresses the bacterial gene for the enzyme xanthine-guanine phosphoribosyltransferase. Mutants deficient in this enzyme can be induced in the AS52 cell line by a variety of mutagens, and spontaneous or induced mutants can be selected for resistance to 6-thioguanine (Tgr). Two Tgr clones derived from the AS52 line were analyzed by Southern blot hybridization and were found to contain deletions involving at least a portion of the gpt gene. Because of the small size and stability of the integrated pSV2gpt plasmid, and the well-defined selection protocol for mutant isolation, the AS52 line offers promise as a system suitable for the study of mutation at the molecular level in CHO cells.We have developed a system to study mutations that affect xanthine-guanine phosphoribosyltransferase (XGPRT) gene (gpt) expression in hypoxanthine-guanine phosphoribosyltransferase-deficient (HGPRT-) CHO cells which have been transformed by the plasmid vector pSV2gpt (10, 11). pSV2gpt carries the Escherichia coli gpt gene and through the use of the simian virus 40 early promoter expresses the bacterial gene for XGPRT in mammalian cells. One isolated CHO transformant, designated AS52, carries a single copy of the gpt gene stably integrated into the high-molecularweight DNA. Bacterial XGPRT and mammalian HGPRT are functionally analogous except that under physiological conditions XGPRT catalyzes the formation of xanthine 5'-monophosphate from xanthine, whereas HGPRT does not. Selection protocols utilizing the purine analog 6-thioguanine for the study of mutation at the hgprt locus in CHO cells (12) can be used to select for mutations at the single gpt gene in the pSV2gpt-transformed CHO line. In this study, we show that mutation induction at the gpt locus in pSV2gpt-transformed CHO cells can be quantified and that specific deletion mutations which affect gpt gene function are the basis for the observed 6-thioguanine-resistant (Tg') phenotype in two mutants derived from this transformed line.The parental line, CHO-K1-BH4 (hgprt+), has been previously described (6). The X3/5 line (HGPRT-), which served as the transformation host for the pSV2gpt gene transfer experiments, was derived from the CHO-K1-BH4 line after an X-ray irradiation of 500 rads and selection in 10 ,uM 6-thioguanine; several lines of evidence indicate that it carries a stable, nonrevertible mutation at the hgprt locus (R. Machanoff, M.S. thesis, University of Tennessee, Knoxville, Tenn., 1982 supplemented with 5% heat-inactivated and dialyzed fetal bovine serum (F12FCM5) at 5% C02, 37°C, and 100%o humidity.The AS52 line is a gpt transformant of X3/5 which carries a single ...

show abstract

“…With EMS we found that mutation induction with a treatment time of 16 hr occurred over the entire survival curve (7,13,14). Further studies using treatment times of 2-24 hr and varying EMS concentrations demonstrated the existence of a limited reciprocity of EMS mutagenesis; that is, when different combinations of EMS concentrations (0.05-3.2 mg/ml) are multiplied by varying treatment times (2-10 hr), the product [(mg/ml)'hr] yields a constant mutation frequency and cytotoxicity (13).…”

Section: Quantitative Mutagenesis With the Cho/hgprt Systemmentioning

confidence: 97%

“…We have routinely used EMS as a positive control for directacting mutagens and found that EMS at 200 ug/ml,for a treatment time of 5 hr consistently yields 300 mutants/10 clonable cells with a 15% standard error (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Several laboratories such as those at DuPont Co., Chemical Industry Institute of Technology, Allied Chemical Co., Carnegie-Mellon Institute, Inhalation Toxicology Research Institute, etc.…”

Section: Mutagen Screening With the Cho/hgprt Systemmentioning

confidence: 99%

“…1, We measure gene mutation by quart ifying the frequency of mutants resistant to a purine analogue, 6-thioguanine (TG). The CHO/HGPRT system has been defined in terms of medium, pH, TG concentration, optimal cell density for selection, recovery of the presumptive mutants, and expression time for the mutant phenotype (6)(7)(8). A metabolic activation system derived from Aroclor 1254-preinduced male Sprague-Dawley rat livers has been used to determine the mutagenicity of promutagens (6,8,9).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Gene Mutation, Quantitative Mutagenesis, and Mutagen Screening in Mammalian Cells: Study with the CHO/HGPRT System

Hsie

1982

Cell Growth

Self Cite

View full text Add to dashboard Cite

SummaryWe have employed CHO cells to develop and define a set of stringent conditions for studying mutation induction to TG resistance. Several lines of evidence support the CHO/HGPRT system as a specific-locus mutatational assay. The system permits quantification of mutation at the HGPRT locus induced by various physical and chemical mutagens. The quantitative nature of the system provides a basis for the study of structure-futvction relationships of various classes of chemical mutagens. The intraand interlaboratory reproducibility of this system suggests its potential for screening environmental agents for mutagenic activity.

show abstract

The dose-response relationship for ethyl methanesulfonate-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells

Cited by 187 publications

References 30 publications

3′ deletions cause aniridia by preventing PAX6 gene expression

3′ deletions cause aniridia by preventing PAX6 gene expression

Detection of deletion mutations in pSV2gpt-transformed cells.

Gene Mutation, Quantitative Mutagenesis, and Mutagen Screening in Mammalian Cells: Study with the CHO/HGPRT System

Contact Info

Product

Resources

About