Sequence analysis of spontaneous mutations in a shuttle vector gene integrated into mammalian chromosomal DNA.

Abstract

We have studied the molecular mechanisms of spontaneous mutations in mouse cells carrying a selectable bacterial gene. The mouse cells carry the Escherichia coli xanthine (guanine) phosphoribosyltransferase (gpt) gene in a retroviral shuttle vector integrated into chromosomal DNA in a proviral form. Cells with spontaneous mutations in the gpt gene were selected as resistant to 6-thioguanine and then were fused with COS cells for recovery of the mutant genes. Out of a total of 77 independent 6-thioguanine-resistant cell lines isolated in this study, vector sequences could be rescued from 43 of the mutant lines, and the base sequences were determined for the gpt genes in all 43 of these lines. There was a variety of mutational events among the mutant gpt genes sequenced. The most frequent mutational event was a deletion (in 29 of the 43 mutant genes), and the next most frequent event was a base substitution mutation (in 11 ofthe 43 mutant genes). Among the deletion mutants, the great majority represent deletions of less than 10 base pairs. In fact, 19 of the 29 deletion mutants had deletions of 3 base pairs, and among the mutants with 3-base. pair deletions, there was a very strong deletion hot spot appearing in 16 independent mutants. Afl 19 of the 3-base-pair deletions resulted in the "in frame" loss of an aspartic acid codon. Among the base substitution mutations, transitions and transversions occurred with approximately equal frequency. Our results raise the possibility that small deletions represent the predominant mechanisms by which spontaneous mutations occur in mammalian cells.Recently, several systems have been described that make possible the rapid and unambiguous determination of base sequence changes in genes that have undergone mutation in mammalian cells (1)(2)(3)(4)(5). In these systems, a target gene is introduced into the mammalian cells as part of a shuttle vector, which is capable of replication in both mammalian cells and bacteria. Following mutagenesis in the mammalian cell environment, the shuttle vector sequences are recovered from the mammalian cells and are introduced into bacteria, where large amounts of the mutant gene can be produced for sequence analysis. The major advantage of these shuttle vector systems is that large numbers of mutant genes can be rapidly isolated in a form that is convenient for DNA sequence analysis.We recently described the development of a system of this type, which utilizes a retroviral shuttle vector (4). As a target gene, this vector contains the Escherichia coli gpt gene, which codes for the enzyme xanthine (guanine) phosphoribosyltransferase (XPRTase). The vector was introduced by infection into the mouse A9 cell line (6), which is deficient in hypoxanithine (guanine) phosphoribosyltransferase. A stable cell line (A9I2), which contains a single copy of the vector integrated into chromosomal DNA in a proviral form, was isolated. Mutants resistant to 6-thioguanine (Sgu) were induced in A9I2 following treatment of the cells with either ethyl methanesu...

Multiple dispersed spontaneous mutations: a novel pathway of mutation in a malignant human cell line.

“…These mutations include all types of changes and appear to fall into two classes: tandem (or nearly tandem, separated by no more than 5 bp) and widely dispersed (separated by hundreds of base pairs). Tandem mutations are rare among spontaneous events analyzed in other systems, accounting for -1% of mutations at the aprt locus of CHO cells (29) and 1 to 2% of mutations of integrated shuttle vector-borne targets in mouse cells (1,18). These mutations can be induced by DNA-damaging agents, such as UV (8,32) or y radiation (24).…”

Use of a selectable marker regulated by alpha interferon to obtain mutations in the signaling pathway.

“…Hygr colonies were generated by cotransfecting 1 ,ug of pSV2hyg and 10 ,ug of pl.8gpt per 5 x 105 cells, using calcium phosphate (20). Forty-eight hours later, the cells were plated in medium containing hygromycin (250 ,ug/ml), and colonies appeared 8 to (6,50,51 7.8], 0.1% bovine serum albumin, 0.1 ,uM KI). Binding buffer (0.6 ml per well) containing labeled IFN-t2 was added, and the plates were rocked gently for 90 min at 20°C.…”

“…The complexity of the genome and the diploid complement of DNA present major obstacles to genetic analyses in mammalian cells. Mutations of a few genes, selected on the basis of resistance to toxic agents, have been used to study the nature of spontaneous or induced mutations in cis (6,9,(49)(50)(51)(52). However, no mutations inactivating factors that stimulate mammalian gene expression in trans have been obtained, even though they would be invaluable in complementing biochemical analyses of the control networks.…”

Disruption of genes regulated during hematopoietic differentiation of mouse embryonic stem cells

“…First, spontaneous HSV-TK inactivation may have been responsible for some false-positive background. Such inactivation has been observed in other systems with frequencies up to 10 -4 and shown to involve both genetic and epigenetic mechanisms (Ashman and Davidson, 1987;GiphartGassler et al, 1989;Tasseron-de Jong et al, 1989) Second, tagged promoters may be silenced because of de novo methylation extending from the provirus into adjacent flanking sequences (Jä hner and Jaenisch, 1985). Third, regulated genes frequently have longer 5Ј-noncoding exons than widely expressed genes (Kozak, 1987), thus providing a larger target capable of activating gene trap expression.…”