Mechanism of mutation at the aprt locus in Chinese hamster ovary cells: analysis of heterozygotes and hemizygotes

1983 ◽  
Vol 3 (10) ◽  
pp. 1703-1710
Author(s):  
A E Simon ◽  
M W Taylor ◽  
W E Bradley
Keyword(s):  
Point Mutation ◽  
High Frequency ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Low Frequency ◽  
Gene Products ◽  
Wild Type ◽  
Gene Copies ◽  
Aprt Gene ◽  
Class 1

A two-step model to explain the high frequency of mutation at the diploid adenine phosphoribosyltransferase (aprt) locus in CHO cells has been proposed previously (Simon et al., Mol. Cell. Biol. 2:1126-1133, 1982). This model indicates that two distinct classes of aprt heterozygotes can be isolated. Class 1 heterozygotes, the most abundant class, were defined as those which arose spontaneously and were capable of undergoing mutation to the APRT- phenotype only at a low frequency (putative point mutation). Class 2 heterozygotes arose from a mutation and gave rise at a high frequency to APRT- cells. This high-frequency event has been identified as a deletion of the wild-type allele (A. E. Simon and M. W. Taylor, Proc. Natl. Acad. Sci. U.S.A. 80:810-814, 1983). In this paper we report further analysis of class 1 heterozygotes with respect to genetic structure, gene products, and karyotype. Our study indicated that class 1 heterozygotes contain two different types of mutants. About half have only one copy of the aprt gene and an unaltered karyotype, indicating that a deletion (similar to the high-frequency second-step event observed for class 2 heterozygotes) rather than a loss of the chromosome was responsible for the generation of the aprt+/- genotype. The remainder of the previously designated class 1 heterozygotes still contained two copies of the aprt gene (within the limits of the quantitation technique used) and arose presumably by a point mutation. One of this group, D423, was characterized with respect to aprt gene products and found to produce an electrophoretic variant in addition to the wild-type protein. APRT- mutants derived from D423 retained the same number of aprt gene copies as D423 and still synthesized a protein that comigrated with wild type, unlike APRT- mutants derived from class 2 heterozygotes. D423 and the other heterozygotes with two aprt genes therefore did not fit into either class 1 or 2 and are now designated class 3. The model we present suggests that only one of the two aprt alleles present in wild-type cells can undergo the deletion.

scholarly journals Mechanism of mutation at the aprt locus in Chinese hamster ovary cells: analysis of heterozygotes and hemizygotes.

1983 ◽  
Vol 3 (10) ◽  
pp. 1703-1710 ◽  
Author(s):  
A E Simon ◽  
M W Taylor ◽  
W E Bradley
Keyword(s):  
Point Mutation ◽  
High Frequency ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Low Frequency ◽  
Gene Products ◽  
Wild Type ◽  
Gene Copies ◽  
Aprt Gene ◽  
Class 1

A two-step model to explain the high frequency of mutation at the diploid adenine phosphoribosyltransferase (aprt) locus in CHO cells has been proposed previously (Simon et al., Mol. Cell. Biol. 2:1126-1133, 1982). This model indicates that two distinct classes of aprt heterozygotes can be isolated. Class 1 heterozygotes, the most abundant class, were defined as those which arose spontaneously and were capable of undergoing mutation to the APRT- phenotype only at a low frequency (putative point mutation). Class 2 heterozygotes arose from a mutation and gave rise at a high frequency to APRT- cells. This high-frequency event has been identified as a deletion of the wild-type allele (A. E. Simon and M. W. Taylor, Proc. Natl. Acad. Sci. U.S.A. 80:810-814, 1983). In this paper we report further analysis of class 1 heterozygotes with respect to genetic structure, gene products, and karyotype. Our study indicated that class 1 heterozygotes contain two different types of mutants. About half have only one copy of the aprt gene and an unaltered karyotype, indicating that a deletion (similar to the high-frequency second-step event observed for class 2 heterozygotes) rather than a loss of the chromosome was responsible for the generation of the aprt+/- genotype. The remainder of the previously designated class 1 heterozygotes still contained two copies of the aprt gene (within the limits of the quantitation technique used) and arose presumably by a point mutation. One of this group, D423, was characterized with respect to aprt gene products and found to produce an electrophoretic variant in addition to the wild-type protein. APRT- mutants derived from D423 retained the same number of aprt gene copies as D423 and still synthesized a protein that comigrated with wild type, unlike APRT- mutants derived from class 2 heterozygotes. D423 and the other heterozygotes with two aprt genes therefore did not fit into either class 1 or 2 and are now designated class 3. The model we present suggests that only one of the two aprt alleles present in wild-type cells can undergo the deletion.

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

1982 ◽  
Vol 2 (9) ◽  
pp. 1126-1133 ◽  
Author(s):  
A E Simon ◽  
M W Taylor ◽  
W E Bradley ◽  
L H Thompson
Keyword(s):  
Chinese Hamster Ovary ◽  
High Frequency ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Low Frequency ◽  
Two Dimensional ◽  
Wild Type ◽  
Proposed Model ◽  
Class 1

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.

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

1982 ◽  
Vol 2 (9) ◽  
pp. 1126-1133
Author(s):  
A E Simon ◽  
M W Taylor ◽  
W E Bradley ◽  
L H Thompson
Keyword(s):  
Chinese Hamster Ovary ◽  
High Frequency ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Low Frequency ◽  
Two Dimensional ◽  
Wild Type ◽  
Proposed Model ◽  
Class 1

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.

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

1983 ◽  
Vol 3 (7) ◽  
pp. 1172-1181
Author(s):  
W E Bradley
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
High Frequency ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Low Frequency ◽  
Class Ii ◽  
Class I ◽  
Wild Type ◽  
Ovary Cells

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.

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

1983 ◽  
Vol 3 (7) ◽  
pp. 1172-1181 ◽  
Author(s):  
W E Bradley
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
High Frequency ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Low Frequency ◽  
Class Ii ◽  
Class I ◽  
Wild Type ◽  
Ovary Cells

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.

scholarly journals Gene Silencing by DNA Methylation and Dual Inheritance in Chinese Hamster Ovary Cells

Genetics ◽  
1998 ◽  
Vol 149 (2) ◽  
pp. 1081-1088
Author(s):  
R P Paulin ◽  
T Ho ◽  
H J Balzer ◽  
R Holliday
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Methylation Pattern ◽  
Pcr Cloning ◽  
Ovary Cells ◽  
Gene Copies ◽  
Aprt Gene ◽  
Dual Inheritance

Abstract Chinese hamster ovary (CHO) cells strain D422, which has one copy of the adenine phosphoribosyl transferase (APRT) gene, were permeabilized by electroporation and treated with 5-methyl deoxycytidine triphosphate. Cells with a silenced APRT gene were selected on 2, 6-diaminopurine. Colonies were isolated and shown to be reactivated to APRT+ by 5-aza-cytidine and by selection in medium containing adenine, aminopterin and thymidine. Genomic DNA was prepared from eight isolates of independent origin and subjected to bisulphite treatment. This deaminates cytosine to uracil in single-stranded DNA but does not deaminate 5-methyl cytosine. PCR, cloning and sequencing revealed the methylation pattern of CpG doublets in the promoter region of the APRT− gene, whereas the active APRT gene had nonmethylated DNA. CHO strain K1, which has two copies of the APRT+ gene, could also be silenced by the same procedure but at a lower frequency. The availability of the 5-methyl dCTP-induced silencing, 5-aza-CR and a standard mutagen, ethyl methane sulphonate, makes it possible to follow concomitantly the inheritance of active, mutant or silenced gene copies. This analysis demonstrates “dual inheritance” at the APRT locus in CHO cells.

Malignancy-related characteristics of wild type and drug-resistant chinese hamster ovary cells

Pathology ◽  
1993 ◽  
Vol 25 (3) ◽  
pp. 268-276 ◽  
Author(s):  
Wanda B. Mackinnon ◽  
Marlen Dyne ◽  
Rebecca Hancock ◽  
Carolyn E. Mountford ◽  
Adrienne J. Grant ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Drug Resistant ◽  
Wild Type ◽  
Ovary Cells

scholarly journals Actin-dependent regulation of the cardiac Na+/Ca2+ exchanger

2006 ◽  
Vol 290 (3) ◽  
pp. C691-C701 ◽  
Author(s):  
Madalina Condrescu ◽  
John P. Reeves
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cytochalasin D ◽  
Lag Phase ◽  
Cell Contact ◽  
Wild Type ◽  
Filamentous Actin ◽  
Surface Distribution ◽  
Reverse Mode ◽  
Hydrophilic Domain

In the present study, the bovine cardiac Na+/Ca2+ exchanger (NCX1.1) was expressed in Chinese hamster ovary cells. The surface distribution of the exchanger protein, externally tagged with the hemagglutinin (HA) epitope, was associated with underlying actin filaments in regions of cell-to-cell contact and also along stress fibers. After we treated cells with cytochalasin D, NCX1.1 protein colocalized with patches of fragmented filamentous actin (F-actin). In contrast, an HA-tagged deletion mutant of NCX1.1 that was missing much of the exchanger's central hydrophilic domain Δ(241–680) did not associate with F-actin. In cells expressing the wild-type exchanger, cytochalasin D inhibited allosteric Ca2+ activation of NCX activity as shown by prolongation of the lag phase of low Ca2+ uptake after initiation of the reverse (i.e., Ca2+ influx) mode of NCX activity. Other agents that perturbed F-actin structure (methyl-β-cyclodextrin, latrunculin B, and jasplakinolide) also increased the duration of the lag phase. In contrast, when reverse-mode activity was initiated after allosteric Ca2+ activation, both cytochalasin D and methyl-β-cyclodextrin (Me-β-CD) stimulated NCX activity by ∼70%. The activity of the Δ(241–680) mutant, which does not require allosteric Ca2+ activation, was also stimulated by cytochalasin D and Me-β-CD. The increased activity after these treatments appeared to reflect an increased amount of exchanger protein at the cell surface. We conclude that wild-type NCX1.1 associates with the F-actin cytoskeleton, probably through interactions involving the exchanger's central hydrophilic domain, and that this association interferes with allosteric Ca2+ activation.

scholarly journals Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter

1994 ◽  
Vol 302 (2) ◽  
pp. 355-361 ◽  
Author(s):  
K Inukai ◽  
T Asano ◽  
H Katagiri ◽  
M Anai ◽  
M Funaki ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Cytochalasin B ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Site Directed Mutagenesis ◽  
Transport Activity ◽  
Wild Type ◽  
In The Wild ◽  
Glut1 Glucose Transporter

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.

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