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Mutants of Chinese hamster ovar cells lacking dihydrofolate reductase (tetrahydrofolate dehydrogenase, 7,8-dihydrofolate:NADP+ oxidoreductase; EC 1.5.1.3) activity were isolated after mutagenesis and exposure to hi -pcificactivity [3H]deoxyuridine as a selective agent. Fully deficient mutants could not be isolated starting with wild-type cells, but could readily be selected from a putative heterozygote that contains half of the wild-type level of dihydrofolate reductase activity. The heterozygote itself was selected from wild-type cells by using [3Hjdeoxyuridine together with methotrexate to reduce intracellular dihydrofolate reductase activity. Fully deficient mutants require glycine, a purine, and thymidine for growth; this phenotype is recessive to wild type in cell hybrids. Revertants have been isolated, one of which produces a heatlabile dihydrofolate reductase activity. These mutants may be useful for metabolic studies relating to cancer chemotherapy and for fine-structure genetic mapping of mutations by using available molecular probes for this gene. (6); (fi) DHFR that is intrinsically less sensitive to MTX (6, 7); or (iii) overproduction of DHFR activity (6,8,9). The last class appears to be the most common, and the overproduction has been shown to result from increased synthesis of wild-type enzyme (10, 11) due to dhfr gene amplification (12). (17) fetal calf serum whenever cell nutrition was being manipulated. Cells were grown at 370C in an atmosphere of 5% carbon dioxide.Selection of DHFR-Deficient Mutants. Mutagenesis with ethyl methanesulfonate (EtMes) and selection of 6-thioguanine-resistant mutants have been described (18). Mutagenesis with y rays was carried out by immersing vials containing cell suspensions in a water tank containing a cobalt-60 source for varying time intervals. The dose used for the isolation of mutants described in Table 2 was 690 rads (1 rad = 1.00 X 10-2 J/kg), which reduced viability to 9%.A partially DHFR-deficient, presumptive

Steady-state dihydrofolate reductase (dhfr) mRNA levels were decreased as a result of nonsense mutations in the dhfr gene. Thirteen DHFR-deficient mutants were isolated after treatment of Chinese hamster ovary cells with UV irradiation. The positions of most point mutations were localized by RNA heteroduplex mapping, the mutated regions were isolated by cloning or by enzymatic amplification, and base changes were determined by DNA sequencing. Two of the mutants suffered large deletions that spanned the entire dhfr gene. The remaining 11 mutations consisted of nine single-base substitutions, one double-base substitution, and one single-base insertion. All of the single-base substitutions took place at the 3' position of a pyrimidine dinucleotide, supporting the idea that UV mutagenesis proceeds through the formation of pyrimidine dimers in mammalian cells. Of the 11 point mutations, 10 resulted in nonsense codons, either directly or by a frameshift, suggesting that the selection method favored a null phenotype. Biol., in press) showed that translation termination mutations in any of the internal exons of the gene gave rise to a low-RNA phenotype, whereas missense mutations in these exons or terminations in exon 6 (the final exon) did not affect dhfr mRNA levels. Nuclear run-on experiments showed that transcription of the mutant genes was normal. The stability of mature dhfr mRNA also was not affected, since (i) decay rates were the same in wild-type and mutant cells after inhibition of RNA synthesis with actinomycin D and (ii) intronless minigene versions of cloned wild-type and nonsense mutant genes were expressed equally after stable transfection. We conclude that RNA processing has been affected by these nonsense mutations and present a model in which both splicing and nuclear transport of an RNA molecule are coupled to its translation. Curiously, the low-RNA mutant phenotype was not exhibited after transfer of the mutant genes, suggesting that the transcripts of transfected genes may be processed differently than are those of their endogenous counterparts.In an effort to identify and understand those aspects of gene structure that play a role in gene expression in mammalian cells, we have been carrying out a detailed mutational analysis of the dihydrofolate reductase (dhfir) gene in Chinese hamster ovary (CHO) cells. dlifr exhibits many typical characteristics of mammalian housekeeping genes: it is 25 kilobase pairs (kbp) in size, contains five variously sized introns, and has a promoter region with no TATA box but with a very high G+C content and an Spl binding site (10,22,40,53). The basic strategy has been to isolate mutants that are deficient in DHFR enzyme activity; such mutants can be readily selected from a CHO cell line that is hemizygous at this locus (58, 59). To focus on lesions that may affect transcription or RNA processing, mutants can be screened for alterations that have affected dimfi-mRNA, either qualitatively or quantitatively.We have previously described spontaneous point mutations in th...

(1) and a corresponding.increase in the gene copy number (2). In some cell lines the amplified genes are lost when the cells are grown in the absence of MTX, whereas in other cell lines the amplified genes are stable. More recently we have found that amplification of H2folate reductase genes occurs in a number of MTX-resistant cell lines derived from both murine and hamster origins, which have stable karyotypes or are highly aneuploid (3). As part of our studies on the mechanism of the amplification, loss, and stabilization of the H2folate reductase genes, it is important to determine the chromosomal localization of these amplified genes.Indirect evidence suggested that there is a specific localization of the H2folate reductase genes. Biedler and Spengler (4) have reported that MTX resistance and elevated reductase levels are associated with a specific chromosomal abnormality in a Chinese hamster lung cell line. In these resistant cells there exists an expanded chromosomal region which displays no banding pattern when stained by the trypsin-Giemsa method, a so-called "homogeneously staining region" (HSR). These workers also reported that in this cell line, in which MTX resistance is unstable, there is a diminution in the average size of the HSR upon loss of MTX resistance (5).We have studied a resistant Chinese hamster ovary (CHO) cell line with H2folate reductase levels and gene copy number approximately 150 times that of sensitive cells. The amplified genes in this cell line are stable, and there exists a characteristic HSR on the second chromosome that is not present in sensitive cells. In situ hybridization of DNA complementary to mRNA of murine H2folate reductase indicates that the reductase genes are specifically localized to the HSR region. MATERIALS AND METHODSIsolation of a MTX-Resistant Variant CHO Clone. Five million unmutagenized CHO-KI cells were exposed to increasing concentrations of MTX in Ham's F12 medium (6) lacking glycine, hypoxanthine, and thymidine, and supplemented with 10% (vol/vol) dialyzed fetal calf serum. At each concentration, cells were cultured until the majority of the culture was growing well; then an inoculum of 5 X 106 cells was used to initiate the next step. The concentrations of MTX used were 0. 01, 0.03, 0.05, 0.07, 0.1, 0.4, 0.8, 1.0, 4.0, 10, 50, and 200

Gamma rays have been used to induce Chinese hamster ovary cell mutants in which the entire locus for dihydrofolate reductase (DHFR) has been eliminated. These mutants were isolated in two steps from a methotrexate-resistant clone (Flintoff, Davidson, and Siminovitch (1976). Somat. Cell Genet. 2, 245-262). The resistant cells contain amplified copies of a mutant dhfr gene that codes for a drug-resistant form of the enzyme. In the first step, methotrexate-sensitive mutants of the amplified line were selected. These mutants exhibit a reduced level of DHFR activity and contain a reduced number of dhfr genes. The remaining DHFR activity is methotrexate-sensitive. These mutants appear to be hemizygotes that have lost all copies of the amplified altered dhfr genes and retain one wild-type allele. In a second mutagenic step, mutants completely deficient in DHFR activity were isolated. Three of four of these mutants were the result of double deletions: they lack all traces of dhfr DNA sequences. The fourth mutant contains a deletion that extends through the 5' half of the dhfr gene. The hemizygotes for dhfr should be useful for the study of mutation at an autosomal mammalian locus without the complications of diploidy.

A series 11 gamma-ray-induced mutants at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary cells has been examined for the types of DNA sequence change brought about by this form of ionizing radiation. All 11 mutants were found to have suffered major structural changes affecting the dhfr gene. In eight of the mutants, all or part of the dhfr gene has been deleted. The extent of these deletions was examined in seven of these mutants and, for comparison, in two deletion mutants that were induced by UV irradiation. For this purpose, probes from an overlapping set of cosmids that span 210 kb of DNA in this region were used. Three of seven gamma-ray-induced mutants and one UV-induced mutant were shown to have deleted the entire 210-kb region. In the remaining mutants, endpoints ranging from within the dhfr gene to 100 kb downstream were observed. No upstream endpoints were detected, so that an upper limit on the size of these large deletions could not be assigned. Three of the 11 gamma-ray-induced mutants contained an interruption in the dhfr gene without any detectable loss of sequence. Restriction analysis of these interrupted mutants showed that at least 8-14 kb of "foreign" DNA sequence became joined to the gene at the point of disruption. Cytogenetic analysis of these mutants showed that in two cases an inversion of the banding pattern on chromosome Z-2 had taken place. The inverted dhfr mutants contain very low amounts of dhfr RNA sequences, and the 5' end of an inversion mutant gene exhibits the same pattern of DNA methylation and DNase I-hypersensitivity as the wild-type gene. Our results suggest that ionizing radiation causes primarily, if not exclusively, large deletions and inversions in mammalian cells.

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease Si analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.Much recent progress has been made toward understanding the mechanism of pre-mRNA splicing in eucaryotic cells, especially since the development of cell-free systems for this process. Analysis of in vitro RNA splicing has led to the discovery of lariat structure splicing intermediates (35,41) and has implicated Ul and U2 small nuclear ribonucleoproteins (snRNPs) as participants in the splicing process (3, 19). The extension of these studies is likely to define the chemical steps involved in the formation of spliced joints. A central problem that remains is how only correctly ordered splices are made in transcripts containing multiple introns, as is the case for the transcripts of most mammalian genes. The evidence on this point is confusing: wild-type transcripts require and exhibit great fidelity in making only the right connections, yet novel splicing can be readily produced experimentally. The alteration of a single base of a splice site can completely prevent splicing at that joint, but new cryptic splice junctions often appear as substitutes (51). It is possible to demonstrate efficient splicing between the exons of two different genes when these genes have been experimentally fused within an intron (12). Finally, splicing can be pro...

Overlapping recombinant lambda 1059 phages carrying regions of the dhfr locus from the amplified Chinese hamster ovary (CHO) cell clone MK42 have been isolated. In addition, dhfr cDNAs from this cell line have been cloned into plasmid pBR322. Restriction analysis of these recombinant molecules has led to a map of the Chinese hamster dhfr gene. This gene has a minimum size of 26 kb and contains six exons as defined by hybridization to a combination of mouse and CHO cDNA probes. The latter probes reveal 3' exonic sequences that are not present in mouse cDNA. The CHO dhfr gene thus extends about 700 bp further 3' than in the mouse, consistent with the larger size of the hamster mRNA. At least five intervening sequences are present, of approximate sizes: 0.3, 2.5, 8.6, 2.6 and 9.4 kb. Four sequences from highly repeated families are situated in introns within the dhfr gene. The overall structure of this gene is strikingly similar to that of the mouse. Evolutionary conservation of interrupted gene structure among mammals thus extends to genes that code for household enzymes as well as specialized or structural proteins.