A mutational hot spot induced by N-hydroxy-aminofluorene in dihydrofolate reductase mutants of Chinese hamster ovary cells.

Eighteen mutants deficient in dihydrofolate reductase (DHFR) activity were induced with 0.5 microM N-hydroxy-aminofluorene in four separate experiments. This carcinogen dose killed approximately 80% of the treated cells and resulted in a mutational frequency approximately 3 x 10(-6). The nature of the induced changes in each of the mutants was determined by direct sequencing following polymerase chain reaction amplification, or in one instance, by Southern blot analysis. Nearly all (15/17) of the mutations were single base changes. Consistent with the binding specificity of this chemical, all mutations were targeted to guanine bases. The predominant change was G:C-->T:A transversion which was evident in 11/15 mutants. A single dG-AF mutational hotspot was noted at a site in the DHFR coding sequence of exon 4; one-third of the induced point mutations arose at this position. These results are compared with our previous analyses of mutants induced with the related aromatic amine, N-2-acetoxy-2-acetyl-aminofluorene.

Splicing mutants and their second-site suppressors at the dihydrofolate reductase locus in Chinese hamster ovary cells.

Point mutants induced with a variety of mutagens at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary (CHO) cells were screened for aberrantly spliced dhfr mRNA by RNase protection and/or reverse transcriptase coupled with cDNA amplification by the polymerase chain reaction (PCR). Of 115 mutants screened, 28 were found to be affected in splicing. All exhibited less than 1% correct splicing, probably because the selection procedure was stringent. All 26 unique mutations were located within the consensus splice sequences; changes were found at 9 of 10 possible sites in this 25-kb six-exon gene. Mutations at the sites flanking the first and last exons resulted in the efficient recruitment of a cryptic site within each exon. In contrast, mutations bordering internal exons caused predominantly exon skipping. In many cases, multiple exons were skipped, suggesting the clustering of adjacent exons prior to actual splicing. Six mutations fell outside the well-conserved GU and AG dinucleotides. All but one were donor site single-base substitutions that decreased the agreement with the consensus and resulted in little or no correct splicing. Starting with five of these donor site mutants, we isolated 31 DHFR+ revertants. Most revertants carried a single-base substitution at a site other than that of the original mutation, and most had only partially regained the ability to splice correctly. The second-site suppression occurred through a variety of mechanisms: (i) a second change within the consensus sequence that produced a better agreement with the consensus; (ii) a change close to but beyond the consensus boundaries, as far as 8 bases upstream in the exon or 28 bases downstream in the intron; (iii) mutations in an apparent pseudo 5' site in the intron, 84 and 88 bases downstream of a donor site; and (iv) mutations that improved the upstream acceptor site of the affected exon. Taken together, these second-site suppressor mutations extend the definition of a splice site beyond the consensus sequence.

Splicing mutations in the CHO DHFR gene preferentially induced by (+/-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene.

Cultured Chinese hamster ovary (CHO) cells were treated with the polycyclic aromatic hydrocarbon racemic 3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene. Mutants deficient in dihydrofolate reductase activity were isolated. A carcinogen treatment at 0.1 microM yielded at 46% survival of the treated population and an induced frequency of mutation of 1.7 x 10(-4), 10(3)-fold greater than the spontaneous rate. By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 38 mutants. Base substitutions accounted for 78% (30/38) of the mutations. We obtained, in addition, four frameshift and four complex mutations. The preferred type of mutation was transversion (A.T----T.A and G.C----T.A) occurring in 69% of the analyzed mutants. A purine was on the 3' side of the putative adduct site in every mutant. Mutations were favored at sequences AGG, CAG, and AAG (the underlined base is the target). Surprisingly, 42% of the mutations created mRNA splicing defects (16/38), especially at splice acceptor sites for each of the five introns. Thus, this chemical carcinogen may recognize some aspect of DNA structure in regions corresponding to pre-mRNA splice sites.

DNA base changes and RNA levels in N-acetoxy-2-acetylaminofluorene-induced dihydrofolate reductase mutants of Chinese hamster ovary cells.

Formerly, we isolated a series of dihydrofolate reductase-deficient Chinese hamster ovary cell mutants that were induced by N-acetoxy-2-acetylaminofluorene. Deletions and complex gene rearrangements were detected in 28% of these mutants; 72% contained putative point mutations. In the present study, we have localized the putative point mutations in the 25,000 base dhfr gene by RNase heteroduplex mapping. Assignment of a position for each mutation was successful in 16 of 19 mutants studied. We cloned DNA fragments containing the mapped mutations from nine mutants into a bacteriophage lambda vector. In the case of 11 other mutants, DNA was amplified by the polymerase chain reaction procedure. Sequence analysis of cloned and amplified DNA confirmed the presence of point mutations. Most mutants (90%) carried base substitutions; the rest contained frameshift mutations. Of the point mutations, 75% were G.C to T.A transversions in either the dhfr coding sequence or at splice sites; transition G.C to A.T mutations were found in two mutants (10%). In one of these transition mutants, the base substitution occurred at the fifth base of the third intron. Of the frameshift mutations, one was a deletion of G.C pair and the other was an insertion of an A.T pair. Of the mapped mutants, 38% exhibited greatly reduced (approximately 10-fold) steady-state levels of dhfr mRNA. All eight sequenced mutants displaying this phenotype contained premature chain termination codons. Normal levels of dhfr mRNA were observed in five missense mutants and in five mutants carrying nonsense codons in the translated portion of exon VI. Taken together with the results of other mutagens at this locus, we conclude that the low dhfr mRNA phenotype is correlated with the presence of nonsense codons in exons II to V but not in the last exon of the dhfr gene.

Nonsense mutations in the dihydrofolate reductase gene affect RNA processing.

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. An examination of steady-state RNA levels in cells carrying these mutations and a comparison with similar data from other dhfr mutants (A. M. Carothers, R. W. Steigerwalt, G. Urlaub, L. A. Chasin, and D. Grunberger, J. Mol. 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.

Point mutation analysis in a mammalian gene: rapid preparation of total RNA, PCR amplification of cDNA, and Taq sequencing by a novel method.

We have developed a very rapid procedure for DNA sequence analysis of induced mutations in a typically large mammalian gene. We are able to determine the nature of chemical carcinogen-induced point mutations in the 25 kb Chinese hamster ovary (CHO) cell dihydrofolate reductase gene within two days starting with 5 to 10 x 10(6) cells. The approach is based on the use of rapidly prepared total RNA from which a 730 bp dhfr cDNA is synthesized by reverse transcriptase and amplified by the polymerase chain reaction (PCR) procedure. Genomic DNA can simultaneously be prepared from the same cells. The amplified double-stranded cDNA is then sequenced directly by the dideoxy method using Taq polymerase in the Thermal Cycler (Perkin Elmer/Cetus). We have previously shown that nonsense codons in the dhfr coding sequence often result in greatly reduced steady-state levels of dhfr mRNA (2). The methods described are suitable for mutants of this type which contain only 1 to 2 copies of mRNA per cell. This approach is readily adaptable to other selectable genes and other cell types provided the necessary primers can be prepared.

Deletion analysis of the Chinese hamster dihydrofolate reductase gene promoter.

Deletion analysis of the 5' flank of the Chinese hamster dihydrofolate reductase (dhfr) gene reveals a promoter region starting 48 base pairs upstream of the major transcriptional start site. A dhfr minigene containing approximately 900 base pairs of 5' flank and one small intron was used as a wild-type standard. Seven deletions were created with BAL-31. Promoter activity was measured in three ways: 1) transient expression of the dhfr gene; 2) frequence of transfection of dhfr- Chinese hamster cells to a dhfr+ phenotype; and 3) RNase protection analysis of dhfr transcripts in pooled populations of permanently transfected cells. The transient expression assay was developed in this work for the rapid analysis of dhfr promoter mutants; this assay could be of general use for analyzing constructs carrying dhfr as a reporter gene. Two of the deletions define a requirement for part or all of the sequence GGGCGT located 48 base pairs upstream of the major transcriptional start site. This site has been shown to bind transcription factor Sp1 in the mouse dhfr gene. The function of the major promoter is independent of the function of the minor promoter. These minigene constructs also contain cryptic promoters located upstream of the natural start sites, probably in the plasmid vector. Transcripts originating from these upstream sites are inefficiently spliced, but do result in messenger RNA molecules that are translated into active dihydrofolate reductase.

Mapping and characterization of mutations induced by benzo[a]pyrene diol epoxide at dihydrofolate reductase locus in CHO cells.

Chinese hamster ovary cells were mutagenized with benzo[a]pyrene diol epoxide (BPDE), an aromatic hydrocarbon carcinogen, and mutants at the dihydrofolate reductase (dhfr) locus were isolated. Of 15 mutants analyzed by Southern blotting, one contained a large deletion that spanned all six exons of the 25-kb dhfr gene; the remaining mutants exhibited no detectable changes. Three of these putative point mutations were localized by the loss of a restriction site: a SacI site in exon III, an MspI site in exon III, and a KpnI site in exon VI. The affected regions in two of these mutants were cloned and sequenced. The SacI- mutant was caused by a G:C----T:A transversion resulting in an amber termination codon. In the MspI- mutant, the deletion of a single C:G resulted in a frameshift and a downstream ochre termination codon. On the basis of overlapping restriction site sequences, the KpnI- mutant was deduced to be a splicing mutant involving the most 3' G in intron V. The location of these and the remaining 11 putative point mutations was sought using RNA heteroduplex mapping. Mismatched bases between riboprobes complementary to wild-type dhfr mRNA and mutant mRNA molecules were detected in 10 of the 14 mutants analyzed. These mutations mapped to four of the six exons or exon splice sites. Surprisingly, over half of these mutants exhibited greatly reduced (approximately 10-fold) steady-state levels of dhfr mRNA.

Polyadenylation of Chinese hamster dihydrofolate reductase genomic genes and minigenes after gene transfer.

The major alternative polyadenylation sites in the Chinese hamster dihydrofolate reductase (dhfr) gene have been identified by DNA sequencing and RNase protection experiments. Comparison of the 3' gene sequence and polyadenylation sites with those of the mouse reveals that, despite an overall sequence homology, the major sites are different in the two species. A series of minigenes was constructed containing the dhfr promoter and the first intron but lacking the four large introns of the genomic sequence. These minigenes contained either all three polyadenylation sites, no polyadenylation sites, or just the first site. All of these minigenes, as well as a cosmid clone containing the full genomic sequence, could transform DHFR-deficient Chinese hamster ovary cell mutants to a DHFR-positive phenotype with approximately equal efficiencies. A minigene lacking the first intron was markedly less efficient. Analysis of dhfr mRNA from transfectant clones derived from minigenes showed that the dhfr polyadenylation sites were used when included, but novel sites were often used in addition. When endogenous polyadenylation sites were absent, new sites in flanking carrier or host DNA were recruited. Transfectants produced by the full genomic dhfr gene yielded mRNA species that were identical in size and relative abundance to the endogenous dhfr gene. The results indicate that the minimal signals for polyadenylation are not complex and can be easily acquired from foreign sequences.

Effect of gamma rays at the dihydrofolate reductase locus: deletions and inversions.

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.

Characterization of mutations induced by 2-(N-acetoxy-N-acetyl)aminofluorene in the dihydrofolate reductase gene of cultured hamster cells.

To determine the types of alterations in gene structure that are induced by the carcinogen 2-(N-acetoxy-N-acetyl)aminofluorene, we used this compound to generate mutations at the dihydrofolate reductase (DHFR) locus (DHFR) in Chinese hamster ovary cells. Twenty-nine independent enzyme-deficient mutants were isolated. A profile of the 26-kilobase (kb)-long gene was obtained by Southern blot analysis of the mutant and parental DNAs digested with BstEII/Kpn I. Hybridization to a mixed probe of 10 DHFR genomic and cDNA fragments revealed 12 bands that scan 34 kb. Twenty-one DHFR- clones (72%) contained small mutations (changes less than 100 base pairs in size). Large or small deletions involving various parts of the gene occurred in eight of the mutants (28%). A large deletion (greater than 35 kb) with 5' and 3' breakpoints mapping to approximately the same location was noted in four mutants. One mutant has undergone a deletion of 550-900 bp that eliminated the first coding exon. Concomitantly, a chromosomal event (either translocation, insertion, or inversion) has separated the 5' flank from the body of the gene. In another mutant, four deletions have occurred at the DHFR 5' end and internally. Restriction fragment length polymorphism analysis of the mutant DNAs with exon-specific probes localized three mutations. One mutant has lost a Taq I (TCGA) site, and another has lost a Sac I (GAGCTC) site. In a third, a GC----TA transversion has created a BstEII (GGTNACC) site. Finally, we used HPLC to determine the ratio of acetylated (12%) to deacetylated (88%) 2-aminofluorene adducts formed in the parental cells. A correlation between the mutational specificities and the conformational changes induced by the two types of DNA adducts is discussed.

Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary 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 S1 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.

Efficient cloning of single-copy genes using specialized cosmid vectors: isolation of mutant dihydrofolate reductase genes.

A method for the efficient cloning of single-copy genes from restriction digests of mammalian DNA is described. The method is illustrated by the cloning of several mutant genes as well as the wild-type gene for Chinese hamster dihydrofolate reductase (DHFR; 7,8-dihydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). This gene is isolated within a 41-kilobase Bgl I fragment by using cosmid (plasmids containing a cohesive-end site) vectors that have been constructed especially for this purpose. Two cosmids are used: one contains a short region from the 5' flanking region of the dhfr gene, and the other contains a short region from the 3' flanking region. These two regions contain the Bgl I sites that bound the dhfr gene. Bgl I leaves staggered ends that are different depending on the DNA sequence within the enzyme binding site. When these cosmids are cut with Bgl I and hybridized with total Bgl I-cut genomic DNA, they preferentially associate with the fragment bearing the dhfr gene, since it has the same Bgl I ends. An approximately 500-fold enrichment for the dhfr gene in cosmid libraries from Chinese hamster ovary cells was achieved by using this method coupled with a single-step size fractionation. As a result, only several hundred cosmid colonies need to be screened in order to clone a dhfr gene from a particular mutant Chinese hamster ovary cell. This method should facilitate the repetitive cloning of any gene or gene fragment.

Use of fluorescence-activated cell sorter to isolate mutant mammalian cells deficient in an internal protein, dihydrofolate reductase.

Dihydrofolate reductase-deficient mutants of Chinese hamster ovary cells have been selected based on their inability to bind a fluorescent derivative of methotrexate, a substrate analog. Nonfluorescent mutant cells were isolated from mutagenized populations using a fluorescence-activated cell sorter. After multiple rounds of sorting plus regrowth, the mutant cell frequency was increased from an initial 10(-5) to greater than 0.9. This use of the cell sorter to isolate mutants deficient in an internal protein should be applicable to any gene product that is able to bind a fluorescent ligand tightly. The method has the advantage of allowing the screening of large numbers of cells and of selecting for partially expressed phenotypes.

Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cells.

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.

Structure of the dihydrofolate reductase gene in Chinese hamster ovary cells.

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.

Selective killing of methotrexate-resistant cells carrying amplified dihydrofolate reductase genes.

A method for the selective killing of methotrexate (MTX)-resistant cells has been developed. The selection is based on the incorporation of tritiated deoxyuridine into the DNA of MTX-resistant cells but not normal MTX-sensitive cells in the presence of the drug. A Chinese hamster ovary cell mutant that overproduces dihydrofolate reductase was used as an example of a MTX-resistant cell line. In this system, a 10,000-fold enrichment for wild-type MTX-sensitive cells could be achieved after 24 hr of exposure to the drug combination. This selection technique was applied to the isolation of MTX-sensitive segregants from hybrid cells formed between the MTX-resistant mutant and wild-type cells. The loss of MTX resistance and dihydrofolate reductase overproduction was always accompanied by the loss of a homogeneously staining region on chromosome 2 of the resistant parent that contains the amplified genes specifying this enzyme. While this region is always lost, other parts of chromosome 2 are almost always retained, suggesting that deletion rather than chromosome loss underlies marker segregation in this case. When the selection was applied to the resistant mutant itself, no MTX-sensitive revertants were obtained among 10(5) cells screened, attesting to the stability of gene amplification in this clone. It is suggested that this combination of drugs may be useful for the elimination of MTX-resistant tumor cells that develop after MTX chemotherapy.

Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity.

Mutants of Chinese hamster ovary cells lacking dihydrofolate reductase (tetrahydrofolate dehydrogenase, 7,8-dihydrofolate:NADP+ oxidoreductase; EC 1.5.1.3) activity were isolated after mutagenesis and exposure to high-specific-activity [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 [3H]deoxyuridine 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 heat-labile 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.