Model involving gene inactivation in the generation of autosomal recessive mutants in mammalian cells in culture.

We present evidence for a two-step model for expression of the recessive phenotype at the diploid adenine phosphoribosyl transferase (aprt) locus in Chinese hamster ovary cells. This model proposes a high-frequency event leading to allelic inactivation and a low-frequency event leading to a structural alteration of the APRT protein. Either event can occur first, resulting in two types of heterozygous cells. The proposed model is based on analysis of Chinese hamster ovary presumptive aprt heterozygotes and APRT- mutants, derived by two different laboratories. The major class of heterozygotes (class 1) had approximately 50% parental APRT activity, 50% immunologically precipitable APRT protein, and only wild-type enzyme as based on two-dimensional gel electrophoresis and thermal inactivation studies. We propose that one allele at the aprt locus has been inactivated in these heterozygotes. APRT- mutants derived from any single class 1 heterozygote arose at a low frequency and contained either no immunologically detectable APRT protein or an APRT enzyme which was, in most cases, demonstrably altered. The second class of heterozygotes, consisting of two independent isolates, gave rise to APRT- cells at a high frequency (10(-3) to 10(-5). These heterozygous cell lines had 50% of parental APRT activity and only wild-type spot, or wild-type and an electrophoretic variant spot, on two-dimensional gels. These aprt heterozygotes appear to have arisen by mutation at one allele. APRT- mutants derived from either heterozygote of this class had all lost the wild-type activity, consistent with the proposed model.     

The aprt heterozygote/hemizygote system for screening mutagenic agents allows detection of large deletions

Frequencies of mutation at the hprt and aprt loci in various CHO cell lines were measured after exposure of the cells to ionizing radiation. In D423 and AA8-16, which are aprt+/- heterozygotes, the ratio of hprt- mutants to aprt- mutants ranged from 0.11 to 0.36. In D422 and AA8-5, which are aprt+/0 cell lines in which only one copy of the gene and its flanking sequences is present these ratios were greater than 5. In contrast, chemical mutagenesis generated mutations at both loci, in all cell lines, at equal frequencies. Southern blot analysis of DNA from hprt- and aprt- mutants of one of the aprt+/- heterozygous lines showed some apparently unaltered genes, some rearrangements and some complete deletions of hprt among hprt- mutants, but only complete deletions of aprt-linked sequences among aprt- mutants. These results strongly suggest that X-ray-induced mutational events are frequently larger than 40 kb (the length of the hprt gene) and that the difference among the frequencies observed at the two loci in the two types of cell lines were due to the presence of essential sequences close the respective target genes. The combined use of these cell lines in screening environmental mutagens should allow qualitative as well as quantitative analysis of the mutagenic potential of environmental agents.     

Correction of a deletion mutant by gene targeting with an adenovirus vector.

The usefulness of adenovirus type 5 as a vector for homologous recombination was examined in CHO cells by using the adenine phosphoribosyltransferase (aprt) gene. Infection of a hemizygous CHO APRT- cell line containing a 3-bp deletion in exon 5 of the aprt gene with a recombinant adenovirus containing the wild-type gene resulted in restoration of the APRT+ phenotype at a frequency of 10(-5) to 10(-6) per infected cell. A relatively high frequency (approximately 6 to 20%) of the transductants appears to result from a homologous recombination event. The mutation on the chromosomal aprt gene is corrected in the homologous recombinants, and APRT expression is restored to a normal hemizygous level. Neither adenovirus nor exogenous promoter sequences are detected in the homologous recombinants. The remaining transductants result from random integration of the aprt gene with the adenovirus sequence. A number of adenovirus vectors containing different promoter sequences linked to the hamster aprt gene were constructed. A possible role for the promoter region in the homologous recombination event was indicated by the lack of homologous recombination in constructs lacking an active promoter.     

Mutation at autosomal loci of Chinese hamster ovary cells: involvement of a high-frequency event silencing two linked alleles.

Two classes of cell lines heterozygous at the galactokinase (glk) locus have been isolated from Chinese hamster ovary cells. Class I, selected by plating nonmutagenized wild-type cells at low density in medium containing 2-deoxygalactose at a partially selective concentration, underwent subsequent mutation to the glk-/- genotype at a low frequency (approximately 10(-6) per cell), which was increased by mutagenesis. Class II heterozygotes, isolated by sib selection from mutagenized wild-type cells, had a higher spontaneous frequency of mutation to the homozygous state (approximately 10(-4) per cell), which was not affected by mutagenesis. About half of the glk-/- mutants derived from a class II heterozygote, but not the heterozygote itself, were functionally hemizygous at the syntenic thymidine kinase (tk) locus. Similarly, a tk+/- heterozygote with characteristics analogous to the class II glk+/- cell lines underwent high-frequency mutation to tk-/-, and most of these mutants, but not the tk+/- heterozygote, were functionally hemizygous at the glk locus. A model is proposed, similar to that for the mutational events at the adenine phosphoribosyl transferase locus (W. E. C. Bradley and D. Letovanec, Somatic Cell Genet. 8:51-66, 1982), of two different events, high and low frequency, being responsible for mutation at either of the linked loci tk and glk. The low-frequency event may be a point mutation, but the high-frequency event, in many instances, involves coordinated inactivation of a portion of a chromosome carrying the two linked alleles. Class II heterozygotes would be generated as a result of a low-frequency event at one allele, and class I heterozygotes would be generated by a high-frequency event. Supporting this model was the demonstration that all class I glk+/- lines examined were functionally hemizygous at tk.     

End extension repair of introduced targeting vectors mediated by homologous recombination in mammalian cells.

We have studied the mechanism of targeted recombination in mammalian cells using a hemizygous adenine phosphoribosyltransferase-deficient (APRT-) Chinese hamster ovary (CHO) cell mutant as a recipient. Three structurally different targeting vectors with a 5' or a 3', or both, end-deleted aprt sequence, in either a closed-circular or linear form, were transfected to the cells with a mutated aprt gene by electroporation. APRT-positive (APRT+) recombinant clones were selected and analyzed to study the gene correction events of the deletion mutation. Some half of 58 recombinant clones obtained resulted from corrections of the deleted chromosomal aprt gene by either gene replacement or gene insertion, a mechanism which is currently accepted for homologous recombination in mammalian cells. However, the chromosomal sequence in the remaining half of the recombinants remained uncorrected but their truncated end of the aprt gene in the incoming vectors was corrected by extending the end beyond the region of homology to the target locus; the corrected vector was then randomly integrated into the genome. This extension, termed end extension repair, was observed with all three vectors used and was as far as 4.6-kilobase (kb) or more long. It is evident that the novel repair reaction mediated by homologous recombination, in addition to gene replacement and gene insertion, is also involved in gene correction events in mammalian cells. We discuss the model which may account for this phenomenon.     

Efficient recruitment of transfected DNA to a homologous chromosomal target in mammalian cells.

A Chinese hamster ovary cell line hemizygous for a defective adenine phosphoribosyltransferase (aprt) gene was transfected with a plasmid, pAG100, capable of correcting the endogenous aprt mutation by targeted homologous recombination. In some experiments, pAG100 was transfected in combination with one of two 'competitor' plasmids. Competitor pCOMP-A was identical to pAG100 except that the aprt sequence on pCOMP-A had the same mutation as the endogenous aprt gene. Competitor pCOMP-B was identical to pAG100 except for a 763 bp deletion in the aprt sequence encompassing the site of mutation in the endogenous gene. Neither pCOMP-A nor pCOMP-B was capable of correcting the defect in the endogenous aprt gene via gene targeting. We asked whether cotransfection of a 4-fold excess of either competitor DNA molecule with pAG100 would reduce the efficiency of targeted correction of the endogenous aprt gene. We report that while plasmid pCOMP-B did not influence the efficiency of gene targeting by pAG100, plasmid pCOMP-A reduced the number of gene targeting events about 5-fold. These observations indicate that the initial homologous interaction between transfected DNA and a genomic target sequence occurs rapidly and that targeting efficiency is limited by a step subsequent to homologous pairing.     

Different capacities for recombination in closely related human lymphoblastoid cell lines with different mutational responses to X-irradiation.

WIL2-NS and TK6 are two distinct human lymphoblast cell lines derived from a single male donor. WIL2-NS cells are significantly more resistant to the cytotoxic effects of X-irradiation but considerably more sensitive to induced mutation. In an effort to determine the mechanistic basis for these differences, we analyzed the physical structures of thymidine kinase (tk)-deficient mutants isolated after X-ray treatment of tk heterozygotes derived from TK6 and the more mutable WIL2-NS. Southern analysis showed that while 84% of TK6-derived mutants had arisen by loss of heterozygosity (LOH), all 106 mutants from WIL2-NS derivatives arose with LOH at tk and all but one showed LOH at other linked loci on chromosome 17. We adapted a fluorescence in situ hybridization technique to distinguish between LOH due to deletion, which results in retention of only one tk allele, and LOH due to a mechanism involving the homologous chromosome (e.g., recombination), which results in the retention of two alleles. Among the LOH mutants derived that were analyzed in this way, 9 of 26 from WIL2-NS and 11 of 17 from TK6 cell lines arose by deletion. The remaining mutants retained two copies of the tk gene and thus arose by a mechanism involving the homologous allele. Since many of these mutants arising by a homologous mechanism retained partial heterozygosity of chromosome 17, they must have arisen by recombination or gene conversion, and not chromosome loss and reduplication. Finally, the recombinational capacities of WIL2-NS and TK6 were compared in transfection assays with plasmid recombination substrates. Intermolecular recombination frequencies were greater in WIL2-NS than in TK6. These data are consistent with a model suggesting that a recombinational repair system is functioning at a higher level in WIL2-NS than in TK6; the greater mutability of the tk locus in WIL2-NS results from more frequent inter- and intramolecular recombination events.     

Isolation and characterization of mutants at the APRT locus in the L-5178Y TK+/TK- mouse lymphoma cell line

2,6-Diaminopurine(DAP)-resistant mutants have been isolated from mouse lymphoma 5178Y TK+/TK- heterozygotes. In the presence of 50 microM DAP, two colony types were isolated. Small colonies contained 50% wild-type adenine phosphoribosyl transferase (APRT) activity (partial mutants), whereas large colonies have undetectable levels of APRT (aprt- mutants). aprt- mutants could be isolated following mutagenesis with ICR-191 or EMS from the partial mutants. Southern blot analysis of EcoRI digested wild-type DNA using a 3.1 kb mouse aprt genomic probe indicated sequence polymorphism at one or both EcoRI sites flanking the allele. Southern blot analysis of one of the partial mutants and one ICR-induced aprt- mutant (single step) indicated that both strains were hemizygous at the APRT locus. Such stable hemizygous strains would be useful in short-term mutagen tests.     

Two novel mutations in the lipoprotein lipase gene in a family with marked hypertriglyceridemia in heterozygous carriers. Potential interaction with the polymorphic marker D1S104 on chromosome 1q21-q23

Two novel mutations in the lipoprotein lipase (LPL) gene are described in an Austrian family: a splice site mutation in intron 1 (3 bp deletion of nucleotides -2 to -4) which results in skipping of exon 2, and a missense mutation in exon 5 which causes an asparagine for histidine substitution in codon 183 and complete loss of enzyme activity. A 5-year-old boy who exhibited all the clinical features of primary hyperchylomicronemia was a compound heterozygote for these two mutations. Nine other family members were investigated: seven were heterozygotes for the splice site mutation, one was a heterozygote for the missense mutation, and one had two wild-type alleles of the LPL gene. LPL activity in the post-heparin plasma of the heterozygotes was reduced to 49;-79% of the mean observed in normal individuals. Two of the heterozygotes had extremely high plasma triglyceride levels; in three of the other heterozygotes the plasma triglycerides were also elevated. As plasma triglycerides in carriers of one defective LPL allele can be normal or elevated, the heterozygotes of this family have been studied for a possible additional cause of the expression of hypertriglyceridemia in these subjects. Body mass index, insulin resistance, mutations in other candidate genes (Asn291Ser and Asp9Asn in the LPL gene, apoE isoforms, polymorphisms in the apoA-II gene and in the apoAI-CIII-AIV gene cluster, and in the IRS-1 gene) could be ruled out as possible factors contributing to the expression of hypertriglyceridemia in this family. A linkage analysis using the allelic marker D1S104 on chromosome 1q21;-q23 suggested that a gene in this region could play a role in the expression of hypertriglyceridemia in the heterozygous carriers of this family, but the evidence was not sufficiently strong to prove this assumption. Nevertheless, this polymorphic marker seems to be a good candidate for further studies.     

Quantitative and molecular analyses of radiation-induced mutation in AS52 cells

pSV2gpt-Transformed and wild-type Chinese hamster ovary (CHO) cell lines have been used to study radiation-induced mutation at the molecular level. The transformant, designated AS52, was constructed from a hypoxanthine-guanine phosphoribosyl transferase (HPRT)-deficient CHO cell line and contains a single, functional copy of the Escherichia coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) stably integrated into the Chinese hamster genome. AS52 and wild-type CHO-K1-BH4 cells exhibit similar cytotoxic responses to uv light and X rays; however, significant differences occur in mutation induction at the gpt and hprt loci. A number of HPRT and XPRT mutants which arose following irradiation were analyzed by Southern-blot hybridization. Most XPRT (21/26) and all HPRT (23/23) mutants induced by uv light exhibited hybridization patterns indistinguishable from their parental cell lines. In contrast, all XPRT (26/26) and most HPRT mutants (15/21) induced by X irradiation contained deletion mutations affecting some or all of the gpt and hprt loci, respectively. These results indicate that X rays induce predominantly deletion mutations, while uv light is likely to induce point mutations at both loci.     

Role of residues 143 and 278 of the human nuclear Vitamin D receptor in the full-length and Delta165-215 deletion mutant

Most of the actions of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] are mediated by binding to the Vitamin D nuclear receptor (VDR). The crystal structure of a deletion mutant (Delta165-215) of the VDR ligand-binding domain (LBD) bound to 1,25(OH)(2)D(3) indicates that amino acid residues tyrosine-143 and serine-278 form hydrogen bonding interactions with the 3-hydroxyl group of 1,25(OH)(2)D(3). Studies of VDR and three mutants (Y143F, S278A, and Y143F/S278A) did not indicate any differences in the binding affinity between the variant receptors and the wild-type receptor. This might indicate that the 3-hydroxyl group binds differently to the full-length VDR than the to deletion mutant. To further investigate, four deletion VDR mutants were constructed: VDR(Delta165-215), VDR(Delta165-215) (Y143F), VDR(Delta165-215) (S278A), VDR(Delta165-215) (Y143F/S278A). There were no significant differences in binding affinity between the wild-type receptor and the deletion mutants except for VDR(Delta165-215) (Y143F/S278A). In gene activation assays, VDR constructs with the single mutation Y143F and the double mutation Y143F/S278A, but not the single mutation S278A required higher doses of 1,25(OH)(2)D(3) for half-maximal response. This suggests that there are some minor structural and functional differences between the wild-type VDR and the Delta165-215 deletion mutant and that Y143 residue is more important for receptor function than residue S278.     

Diploids heterozygous for a vma13Delta mutation in Saccharomyces cerevisiae highlight the importance of V-ATPase subunit balance in supporting vacuolar acidification and silencing cytosolic V1-ATPase activity

The V-ATPase H subunit (encoded by the VMA13 gene) activates ATP-driven proton pumping in intact V-ATPase complexes and inhibits MgATPase activity in cytosolic V1 sectors (Parra, K. J., Keenan, K. L., and Kane, P. M. (2000) J. Biol. Chem. 275, 21761-21767). Yeast diploids heterozygous for a vma13Delta mutation show the pH- and calcium-dependent conditional lethality characteristic of mutants lacking V-ATPase activity, although they still contain one wild-type copy of VMA13. Vacuolar vesicles from this strain have approximately 50% of the ATPase activity of those from a wild-type diploid but do not support formation of a proton gradient. Compound heterozygotes with a second heterozygous deletion in another V1 subunit gene exhibit improved growth, vacuolar acidification, and ATP-driven proton transport in vacuolar vesicles. In contrast, compound heterozygotes with a second deletion in a Vo subunit grow even more poorly than the vma13Delta heterozygote, have very little vacuolar acidification, and have very low levels of V-ATPase subunits in isolated vacuoles. In addition, cytosolic V1 sectors from this strain and from the strain containing only the heterozygous vma13Delta mutation have elevated MgATPase activity. The results suggest that balancing levels of subunit H with those of other V-ATPase subunits is critical, both for allowing organelle acidification and for preventing unproductive hydrolysis of cytosolic ATP.     

Structure and sequence of mutations induced by ionizing radiation at selectable loci in Chinese hamster ovary cells

The spectrum of mutations induced by ionizing radiation at two non-essential genetic loci varies markedly. Those at the adenine phosphoribosyl transferase (aprt) locus predominantly have no detectable alterations of gene structure on Southern blots, while those at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus are largely massive deletions eliminating all coding sequence. Insertion mutations were detected at both loci. To characterize the sequence alterations producing the minor changes at the aprt locus, two mutant genes were cloned from lambda genomic libraries and sequenced. One of these mutants proved to be a 20 base-pair deletion formed between two short (3 base-pair) direct repeat sequences, while the second was the result of a 58 base-pair insertion accompanied by a 13 base-pair deletion.     

Spontaneous and ionizing radiation induced mutations involve large events when selecting for loss of an autosomal locus

The mouse P19H22 embryonal carcinoma cell line contains two distinct chromosome 8 homologs, one derived from Mus musculus domesticus (M. domesticus) and the other derived from Mus musculus musculus (M. musculus). It also contains a deletion for the M. musculus aprt allele, which is located on chromosome 8. In this study, cells with spontaneous or induced aprt deficiencies were isolated from P19H22 and examined to determine the nature of the mutational events that had occurred. Ultraviolet radiation (UV), ethyl methanesulfonate (EMS), and two forms of ionizing radiation, ¹³⁷Cs and ²⁵²Cf, were used for mutation induction. DNA preparations from the aprt deficient cells were initially screened with a Southern blot analysis and separated into two broad classes: those that had lost the M. domesticus aprt allele and those that had retained it. The overwhelming majority ( > 95%) of the spontaneous and ionizing radiation-induced mutants exhibited aprt gene loss, indicating that relatively large events had occurred and that homozygosity for the deleted region was not a lethal event. Loss of heterozygosity for syntenic markers was found to be a common event in cells exhibiting aprt gene loss. In contrast, a majority of the UV-induced mutants (61%) and a substantial minority of the EMS-induced mutants (38%) retained the aprt gene. A sequence analysis confirmed that base-pair substitutions were responsible for this class of mutation. Gene inactivation associated with hypermethylation of the promoter region was found to be a rare event and was not induced by any of the mutagenic agents tested. The results demonstrate the suitability of the P19H22 cell line for mutational studies, particularly those that are large in nature.     

Structural alterations of the aprt locus induced by deoxyribonucleoside triphosphate pool imbalances in Chinese hamster ovary cells.

Mutants induced at the adenine phosphoribosyl transferase (aprt) locus by dTTP or dCTP pool imbalances were examined for alterations in genomic DNA sequences. No observable changes were detected by Southern blot analysis of most mutant DNAs, suggesting induction of base pair alterations or other events below our level of detection (approximately 30 base pairs). However, in a few strains (11 from a total collection of 125 mutant cell strains), we were able to localize these events to restriction endonuclease recognition sequences when the mutations resulted in the loss or gain of a particular site. The distribution of lost or gained sites in aprt-deficient mutants induced by the two types of pool imbalances clearly varied, with those occurring in a mutator strain with increased dCTP clustering at one end of the aprt gene. Mutants induced by dTTP also revealed novel events: multiple restriction site modifications in a small region of the aprt gene in one mutant and a small (approximately 50 base pairs) insertion or duplication of DNA sequences. As in previous studies, very few deletion or insertion mutants were detected at the aprt locus. The significance of these findings in terms of the known biochemical and genetic consequences of these pool imbalances is discussed.     

DNA amplification--deletion in a spontaneous mutation of the hamster aprt locus: structure and sequence of the novel joint.

In a collection of spontaneous mutants of Chinese hamster ovary cells selected for deficiency in adenine phosphoribosyl transferase (aprt) activity, one was detected having not only a deletion of aprt coding sequences but also an apparent amplification of remaining sequences. The HindIII fragment bearing the novel joint was cloned and sequenced revealing a complex gene rearrangement. A deletion of at least 9 kb extending upstream from the aprt locus is accompanied by an inverted duplication of flanking sequences 672 bp downstream from the novel joint. This unit is amplified three to four times with the net result of some sequences being increased as much as eight fold in copy number because of the duplication. The fidelity of the sequences involved is preserved. We propose a model which could account for this inverted duplication.     

Southern Analysis of Genomic Alterations in Gamma-Ray-Induced Aprt- Hamster Cell Mutants

The role of genomic alterations in mutagenesis induced by ionizing radiation has been the subject of considerable speculation. By Southern blotting analysis we show here that 9 of 55 (approximately 1/6) gamma-ray-induced mutants at the adenine phosphoribosyl transferase (aprt) locus of Chinese hamster ovary (CHO) cells have a detectable genomic rearrangement. These fall into two classes: intragenic deletions and chromosomal rearrangements. In contrast, no major genomic alterations were detected among 67 spontaneous mutants, although two restriction site loss events were observed. Three gamma-ray-induced mutants were found to be intragenic deletions; all may have identical break-points. The remaining six gamma-ray-induced mutants demonstrating a genomic alteration appear to be the result of chromosomal rearrangements, possibly translocation or inversion events. None of the remaining gamma-ray-induced mutants showed any observable alteration in blotting pattern indicating a substantial role for point mutation in gamma-ray-induced mutagenesis at the aprt locus.     

Charcot-Marie-Tooth-linked mutant GARS is toxic to peripheral neurons independent of wild-type GARS levels

Charcot-Marie-Tooth disease type 2D (CMT2D) is a dominantly inherited peripheral neuropathy caused by missense mutations in the glycyl-tRNA synthetase gene (GARS). In addition to GARS, mutations in three other tRNA synthetase genes cause similar neuropathies, although the underlying mechanisms are not fully understood. To address this, we generated transgenic mice that ubiquitously over-express wild-type GARS and crossed them to two dominant mouse models of CMT2D to distinguish loss-of-function and gain-of-function mechanisms. Over-expression of wild-type GARS does not improve the neuropathy phenotype in heterozygous Gars mutant mice, as determined by histological, functional, and behavioral tests. Transgenic GARS is able to rescue a pathological point mutation as a homozygote or in complementation tests with a Gars null allele, demonstrating the functionality of the transgene and revealing a recessive loss-of-function component of the point mutation. Missense mutations as transgene-rescued homozygotes or compound heterozygotes have a more severe neuropathy than heterozygotes, indicating that increased dosage of the disease-causing alleles results in a more severe neurological phenotype, even in the presence of a wild-type transgene. We conclude that, although missense mutations of Gars may cause some loss of function, the dominant neuropathy phenotype observed in mice is caused by a dose-dependent gain of function that is not mitigated by over-expression of functional wild-type protein.