Gail Urlaub

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 dhfrgene. In eight of the mutants, all or part of the dhfrgene 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 dhfrgene 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 dhfrgene 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 dhfrmutants contain very low amounts of dhfrRNA 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.

Mutant alleles for hypoxanthine phosphoribosyltransferase: Codominant expression, complementation, and segregation in hybrid Chinese hamster cells

Chinese hamster ovary cell mutants resistant to the purine analogs 6-thioguanine or 8-azaguanine have been isolated following mutagenesis with ethyl methane sulfonate. The activities of hypoxanthine phosphoribosyl-transferase (HPRT) in three such mutants have been found to exhibit an increased Kmfor the substrate 5-phosphoribosyl-1-pyrophosphate. The isoelectric point of the mutant enzyme activity has also changed in two mutants. Hybrid cells containing one mutant and one wild-type allele express both genes. Segregants that have lost only the wild-type allele can be selected on the basis of drug resistance. Two mutants exhibiting different alterations in HPRT activity can complement in a hybrid cell to yield a wild-type growth pattern and enzyme activity with intermediate electrophoretic and kinetic properties. The results suggest intracistronic complementation between structural gene mutants of HPRT.

Transformation of the gene for hypoxanthine phosphoribosyltransferase.

Purified DNA from wild-type Chinese ovary (CHO) cells has been used to transform three hypoxanthine phosphoribosyltransferase (HPRT) deficient murine cell mutants to the enzyme positive state. Transformants appeared at an overall frequency of 5×10−8 colonies/treated cell and expressed CHO HPRT activity as determined by electrophoresis. One gene recipient, B21, was a newly isolated mutant of LMTK− deficient in both HPRT and thymidine kinase (TK) activities. Transformation of B21 to HPRT+ occurred at 1/5 the frequency of transformation to TK+; the latter was, in turn, an order of magnitude lower than that found in the parental LMTK− cells, 3×10−6. Thus both clonal and marker-specific factors play a role in determining transformability. The specific activity of HPRT in transformant extracts ranged from 0.5 to 5 times the CHO level. The rate of loss of the transformant HPRT+ phenotype, as measured by fluctuation analysis, was 10−4/cell/generation. While this value indicates stability compared to many gene transferents, it is much greater than the spontaneous mutation rate at the indigenous locus. The ability to transfer the gene for HPRT into cultured mammalian cells may prove useful for mutational and genetic mapping studies in this well-studied system.

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.

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-kbdhfr 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-typedhfr 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 ofdhfr mRNA.

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.

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 dhfrpromoter 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 dhfrmRNA from transfectant clones derived from minigenes showed that the dhfrpolyadenylation 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 dhfrgene yielded mRNA species that were identical in size and relative abundance to the endogenous dhfrgene. The results indicate that the minimal signals for polyadenylation are not complex and can be easily acquired from foreign sequences.

[34] Repetitive cloning of mutant genes using locus-specific sticky ends

Publisher Summary This chapter describes method of preparing a recombinant DNA library that is enriched for one particular gene sequence. This method involves the use of vectors containing small regions derived from the flanks of the wild-type gene. The vectors are cut in such a way as to produce sticky ends that will preferentially ligate to the ends of the fragment to be cloned. The method described in the chapter presents an alternative to methods based on homologous recombination in E. coli . In comparison with those procedures, it provides some potential advantages. No special bacterial hosts are required. The use of recA hosts minimizes the chances that a cloned gene may be altered by the cloning regimen. The cloning sites precisely define the extent of the fragment that has been cloned. Finally, in principle it is not restricted to phage-based vectors.