An adenosine kinase mutation in baby hamster kidney cells causing increased sensitivity to adenosine

A class of aravinosyladenine (araA)-resistant mutants of baby hamster kidney (BHK 21/C13) cells exhibits multiple phenotypes: resistance to araA and deoxyadenosine, extreme sensitivity to adenosine (Ado) and varying degrees of deficiency in adenosine kinase (AK) activity. One of these Ado^s/araA^r strains, ara-S10d, was isolated without mutagenesis and was shown to possess about 59% level of the wild-type AK activity. The AK from ara-S10d had an altered K_m and pH optimum and was stimulated by K⁺ cations. A number of Ado^s to Ado^r revertants were isolated from araS10d, and in all of the 7 examined, the AK activity was reduced to a nondetectable level. The altered kinetic parameters of the AK enzyme in ara-S10d cells suggest a mutation of the AK gene that leads to the synthesis of an altered enzyme. The loss of AK activity in the Ado^r revertants suggests an association of the enhanced Ado sensitivity to the AK mutation.     

Inhibition of methylation of DNA and tRNA by adenosine in an adenosine-sensitive mutant of the baby hamster kidney cell line

An adenosine-sensitive (Ados) mutant of baby hamster kidney (BHK) cells, ara-S10d, when treated with a toxic concentration of adenosine (Ado), displayed a substantial elevation of S-adenosylhomocysteine (SAH), S-adenosylmethionine (SAM), and methylthioadenosine (MTA). Wild-type BHK cells treated with the same concentration of Ado (not toxic to these parental cells) produced an elevation of SAH 1.5 times higher than that of ara-S10d cells without a concurrent elevation of SAM or MTA. Inhibition of methylation of DNA and tRNA is greater in ara-S10d cells treated with Ado than that of similarly treated wild-type cells. This inhibition was correlated with the enhanced Ado toxicity, suggesting inhibition of methylation as a possible causal factor for the great increase in Ado sensitivity. Inhibition of methylation may be due to the elevated level of MTA and not solely to the elevation of SAH, a well-known potent inhibitor of numerous methyltransferases.     

Codominant translational mutants of Chinese hamster ovary cells selected with diphtheria toxin.

Diphtheria toxin-resistance markers in two translational mutants, CH-RE1.22c, possessing no toxin-sensitive EF-2 (class IIa), and CH-RE1.32, with 50% toxin-sensitive and 50% toxin-resistant EF-2 (class IIb), behaved codominantly in somatic cell hybrids. There was no complementation in hybrids formed between the two resistant mutants. The mutant parents and their hybrids, except those formed by fusion of CH-RE1.32 and wild-type cells, grew in the presence of toxin. To explain these results we suggest that CHO-K1 cells possess two functional copies of the gene for EF-2 and that CH-RE1.22c and CH-RE1.32 represent the homozygous (R/R) and heterozygous (R/S) states of resistance at the EF-2 gene locus. The failure of hybrids formed between CH-RE1.32 and wild-type cells to grow in toxin is a gene dosage effect. Codominant class IIa translational resistance is a selectable marker for the isolation of hybrids. It can be combined with a second, recessive, marker to provide a cell which is a "universal hybridizer" (10).     

Six complementation groups for ionising-radiation sensitivity in Chinese hamster cells

The ionising radiation-sensitive mutants irs 1, irs 2, irs 3, xrs-1 (or xrs-7), EM7 and XR-1 were fused to wild-type cells or to each other in pairs to create hybrid cells. These hybrids were checked chromosomally and their X-ray sensitivity tested. Each mutant was found to be recessive to wild-type and to complement the X-ray sensitivity of the other mutants. Thus there appear to be at least 6 complementation groups for ionising radiation sensitivity in Chinese hamster cells.     

Identification of the CysB-regulated gene,hslJ, related to the Escherichia coli novobiocin resistance phenotype

The cysB gene product is a LysR-type regulatory protein required for expression of the cys regulon. cysB mutants of Escherichia coli and Salmonella, along with being auxotrophs for the cysteine, exhibit increased resistance to the antibiotics novobiocin (Nov) and mecillinam. In this work, by using lambdaplacMu9 insertions creating random lacZ fusions, we identify a gene, hslJ, whose expression appeared to be increased in cysB mutants and needed for Nov resistance. Measurements of the HSLJ::lacZ gene fusion expression demonstrated that the hslJ gene is negatively regulated by CysB. In addition we observe the negative autogenous control of HslJ. When the control imposed by CysB is lifted in the cysB mutant, the elevation of Nov resistance can be achieved only in the presence of wild-type hslJ allele. A double cysB hslJ mutant restores the sensitivity to Nov. Overexpression of the wild-type HslJ protein either in a cysB(+) or a cysB(-) background increases the level of Nov resistance indicating that hslJ product is indeed involved in accomplishing this phenotype. The HSLJ::OmegaKan allele encodes the C-terminaly truncated mutant protein HslJ Q121Ter which is not functional in achieving the Nov resistance but when overexpressed induces the psp operon. Finally, we found that inactivation of hslJ does not affect the increased resistance to mecillinam in cysB mutants.     

Reduction to homozygosity is the predominant spontaneous mutational event in cultured human lymphoblastoid cells

Expression of recessive mutant phenotypes can occur by a number of different mechanisms. Inactivation of the wild-type allele by base-substitution mutations, frameshift mutations or small deletions occurs at both hemizygous and heterozygous cellular loci, while other events, such as chromosome level rearrangements, may not be detected at hemizygous loci because of inviabiltty of the resulting mutants. In order to assess the relative contribution of each type of mutational event, we isolated a human lymphoblastoid cell line that is heterozygous at the adenine phosphoribosyltransgerase (aprt) locus. The mutation rate for the expression of the mutant phenotype (aprt^+/−→aprt^−/−) was 1.3 × 10⁻⁵/cell/ generation. Molecular analysis of the DNA from 26 mutant clones revealed that 19% had undergone deletion of the entire wild-type allele. The aprt heterozygote carries a mutation in the coding sequence of the gene that results in the loss of a restriction site. Analysis of aprt^−/− mutants for this restriction fragment length difference reveales that 23% of the mutants contained point mutations or small ((< 100 bp) deletions. The remainder of the mutants (58%) resulted from reduction to homozygosity of the mutant allele. We suggest that, as in tumor cells in vivo, reduction to homozygosity is a major mechanism for the expression of recessive mutant phenotypes in cultured human cells.     

Genetic and biochemical properties of thialysine-resistant mutants of Saccharomyces cerevisiae

Three groups of lysine-excreting, thialysine-resistant mutants of Saccharomyces cerevisiae were derived from the wild-type strain (X2180) by mutagenic treatment and selected on the basis of a cross-feeding assay. Mutants MNNG2-9, MNNG2-27, MNNG2-39 and MNNG2-62 (group 1) exhibited a 2:2 segregation for thialysine resistance following mating with a wild-type strain and a lower than wild-type lysyl-tRNA synthetase activity; the thialysine-resistant phenotype was dominant in specific hybrids. Mutant MNNG2-2 (group II) was similar to group I mutants except that the thialysine-resistant phenotype was recessive in the hybrid. Mutant MNNG3-142 (group III) exhibited an irregular ratio of segregation of thialysine resistance and a significantly lower lysyl-tRNA synthetase activity; the thialysine-resistant phenotype was recessive in the hybrid. The growth of both group I and group III mutants was temperature-sensitive. The thialysine-resistant mutants exhibited pleiotropic properties including the increased production and excretion of lysine, thermosensitive growth and an impairment of lysyl-tRNA synthetase activity.     

Formycin B-resistant mutants of Chinese hamster ovary cells: novel genetic and biochemical phenotype affecting adenosine kinase.

Stable mutants which are approximately three- and eightfold resistant to the pyrazolopyrimidine nucleosides formycin A and formycin B (FomR) have been selected in a single step from mutagenized Chinese hamster ovary cells. In cell extracts, the two FomR mutants which were examined were both found to contain no measurable activity of the enzyme adenosine kinase (AK). However, cross-resistance studies with other adenosine analogs such as toyocamycin and tubercidin show that these mutants are distinct from toyocamycin or tubercidin resistant (Toyr) mutants which also contain no measurable AK activity in cell extracts. Studies on the uptake and incorporation of [3H]adenosine and [3H]tubercidin by various mutants and parental cell lines show that unlike the Toyr mutants, which are severely deficient in the phosphorylation of these compounds, the FomR mutants possess nearly normal capacity to phosphorylate these compounds and incorporate them into cellular macromolecules. These results suggest that the FomR mutants contain normal levels of AK activity in vivo. In cell hybrids formed between FomR X FomS cells and FomR X Toyr cells, the formycin-resistant phenotype of both of the FomR mutants behaved codominantly. However, the extracts from these hybrid cells contained either congruent to 50% (FomR X FomS) or no measurable (FomR X Toyr) AK activity, indicating that the lesion in these mutants neither suppresses the wild-type AK activity nor complements the AK deficiency of the Toyr mutants. The presence of AK activity in the FomR mutants in vivo, but not in their cell extracts, along with the codominant behavior of the mutants in hybrids, indicates that the lesions in the FomR mutant are of a novel nature. It is suggested that the genetic lesion in these mutants affects AK activity indirectly and that it may involve an essential cellular function which exists in a complex form with AK. Some implications of these results regarding the mechanism of action of formycin B are discussed.     

High frequency of mutation to tubercidin resistance in CHO cells.

The acquisition of high-level resistance to tubercidin (an adenosine analog) in CHO cells occurs in a single step at high frequency (10(-3) to 10(-4)) without mutagenesis. Analysis of a large number of independent mutants by a fluctuation test (Luria and Delbruk, 1943) indicates that they arise independently of the selection medium and all fall into the same complementation group. All mutants tested lack detectable adenosine kinase activity. An analysis of hybrids between mutant and wild-type cells indicates that resistance to tubercidin is a recessive marker which segregates as would be expected if it were a haploid locus in the parental CHO cell. Resistance to tubercidin is not linked to the X chromosome in CHO cells and appears to occur at much lower frequency in primary Chinese hamster cells and other cultured cell lines.     

The genetic basis of resistance to ionising radiation damage in cultured mammalian cells

To test the genetic similarity of independently-isolated hamster cell mutants sensitive to ionising radiation, these were fused in pairs and the hybrids exposed to X-rays. Some mutants (irs1, irs3, xrs-1, XR-1, BLM2) were found to complement all others tested for radiosensitivity in hybrids, and are therefore in separate genetic groups. The mutants irs2 and V-E5, both isolated from V79 cells, did not complement and therefore belong to the same group. Another pair, EM7 and irs1SF, formed hybrids with intermediate levels of survival between mutant and wild-type. However, the parental cells fused to irs1SF also showed intermediate sensitivity, suggesting a semi-dominant mutant phenotype rather than a lack of complementation. Crosses of some of these hamster mutants to the radiosensitive mouse mutant M10 showed clear complementation (irs1 x M10, irs2 x M10) but for others the complementation did not greatly exceed the sensitivity of one (irs3 x M10) or both mutants (XR-1 x M10). Taken with our previously-published data, these results show that there are at least 8 genetic groups determining resistance to ionising radiation damage in rodent cells.     

Genetic analysis of mitomycin-C-sensitive mutants of a Chinese hamster ovary cell line

5 mutants of a Chinese hamster ovary (CHO) cell line, which exhibit similar levels of sensitivity to killing by mitomycin C, have been analysed genetically to determine whether they represent one or more genetic complementation groups. Hybrids were constructed by fusing cells carrying either the neo or the Ecogpt marker and selecting in medium containing G418 and mycophenolic acid. Selectable markers were introduced into the cells by DNA transfection using pSV5-neo or pSV5-gpt, which represents a quick and convenient method for generating resistant derivatives. Hybrids generated by crosses between any one mutant and the parental cell line exhibited near wild-type resistance to mitomycin C, indicating that the mutants are phenotypically recessive. Self-cross hybrids for all 5 mutants had D37 values for killing by mitomycin C of between 20 and 30 ng/ml. The values obtained for crosses between different mutants were 60-105 ng/ml, with the exception of 1 pairing which gave a value of 33 ng/ml. These results indicate that that the mutants represent at least 4 different genetic complementation groups, suggesting that cellular resistance to mitomycin C is mediated via a number of different mechanisms.     

Ribosomal and non-ribosomal resistance to oxazolidinones: species-specific idiosyncrasy of ribosomal alterations

A derivative of Mycobacterium smegmatis, which carries only one functional rRNA (rrn) operon, was used to isolate mutants resistant to the ribosome-targeted antibiotic linezolid. Isolation and characterization of linezolid-resistant clones revealed two classes of mutants. Ribosomes from class I mutants are resistant to oxazolidinones in an in vitro peptidyl transferase assay, indicating that resistance maps to the ribosome component. In contrast, ribosomes from class II mutants show wild-type susceptibility to a linezolid derivative in vitro, pointing to a non-ribosomal mechanism of resistance. Introduction of a wild-type ribosomal RNA operon into linezolid-resistant strains restored linezolid sensitivity in class I mutants, indicating that resistance (i) maps to the rRNA and (ii) is recessive. Sequencing of the entire rrn operon identified a single nucleotide alteration in 23S rRNA of class I mutant strains, 2447G --> T (Escherichia coli numbering). Introduction of mutant rrl2447T into M. smegmatis rrn- resulted in a linezolid-resistant phenotype, demonstrating a cause-effect relationship of the 2447G --> T alteration. The 2447G --> T mutation, which renders M. smegmatis linezolid resistant, confers lethality in E. coli. This finding is strong evidence of structural and pos-sibly functional differences between the ribosomes of Gram-positive and Gram-negative bacteria. In agreement with the results of the in vitro assay, class II mutants show a wild-type sequence of the complete rRNA operon. The lack of cross-resistance of the class II mutants to other antibiotics suggests a resistance mechanism other than activation of a broad-spectrum multidrug transporter.     

Responsiveness to transforming growth factor-beta (TGF-beta) restored by genetic complementation between cells defective in TGF-beta receptors I and II.

Selection of mutant Mv1Lu mink lung epithelial cells resistant to growth inhibition by transforming growth factor-beta (TGF-beta) has led to the isolation of cell clones with distinct alterations in type I and II TGF-beta receptors. Certain mutant clones present a decreased number or complete loss of detectable type I receptor. Other clones show a loss and/or altered electrophoretic mobility of the type II receptor, with concomitant loss of the type I receptor. Using somatic cell hybridization analysis we demonstrate the recessive nature of these mutants with respect to the wild-type phenotype and define various mutant complementation groups. Among these, hybrids between cells that express only type II receptor (R mutants) and cells that express neither receptor type (DRa mutants) rescue wild-type expression of type I receptors. Moreover, these hybrids regain full responsiveness to TGF-beta 1, as measured by inhibition of DNA synthesis as well as stimulation of fibronectin and plasminogen activator inhibitor-1 production. These results provide evidence for an interaction between TGF-beta receptor components I and II and show that, in Mv1Lu cells, expression of both receptor types is required for mediation of biological responses to TGF-beta 1.     

The molecular basis of emetine resistance in chinese hamster ovary cells: Alteration in the 40S ribosomal subunit

The molecular basis of resistance to the protein synthesis inhibitor emetine has been examined in cell-free, protein-synthesizing extracts derived from normal and emetine-resistant (Emt^R) mutants. We had earlier shown that protein synthesis in extracts of the mutant cells was resistant to the inhibitory action of the emetin. When extracts from a wild-type and mutant cell line were fractionated into supernatant (S-100) and polyribosome fractions and mixed in different combinations, resistance to emetine was found to be associated with the mutant polyribosome fraction. Further fractionation of wild-type and mutant polyribosomes into 40S and 60S ribosomal subunits and mixing them in various combinations with an S-100 fraction from the wild-type cell indicates that resistance of mutant cells to emetine involves an alteration in the 40S ribosomal subunit.The behavior of Emt^R has also been examined in somatic cell hybrids. Studies of Emt^R × Emt^S hybrid cell lines in vivo and in vitro show that Emt^R is phenotypically recessive to Emt^S, which is consistent with the ribosomal location of the genetic change.     

Gene inactivation as a mechanism for the expression of recessive phenotypes.

A series of Chinese hamster ovary cell hybrids were constructed which were heterozygous at the emtB and chr loci. These loci encode two recessive drug-resistance genes (emetine resistance and chromate resistance, respectively) located on a structurally hemizygous region on the long arm of chromosome 2. These heterozygous hybrids therefore exhibit wild-type sensitivity to both emetine and chromate. Drug-resistant variants were then selected in medium containing either emetine or chromate, and the mechanism of reexpression of the recessive drug-resistant allele was determined by karyotypic analysis of the resultant colonies. In previous studies at these loci we have determined that segregation of the recessive phenotype occurs primarily by (1) the loss of the chromosome 2 carrying the wild-type, drug-sensitive, allele, (2) deletion of the long arm of chromosome 2, or (3) loss of one chromosome 2 followed by duplication of the remaining homologue. However, a small proportion of segregants have also been detected which may have arisen by the mechanisms of de novo gene inactivation or mutation. In this report, hybrids are described which were constructed to allow selection for the retention of the chromosome carrying the wild-type allele and which therefore optimize isolation of these rare segregants. We demonstrate by karyotypic analysis, mutation frequency analysis, and microcell-mediated chromosome transfer that these rare segregants occur primarily by gene inactivation. We also demonstrate a dramatic increase in the proportion of segregants occurring by gene inactivation in two of these hybrids as compared with those previously reported, indicating that this mechanism may be an important mode of phenotype segregation in diploid cells and, therefore, in the development of cancers--such as the childhood tumors retinoblastoma and Wilms tumor--resulting from recessive alleles     

Complementation analyses of differentiation-defective embryonal carcinoma cells

We have generated cell hybrids by fusing embryonal carcinoma (EC) cells which fail to differentiate in response to retinoic acid (RA) and/or hexamethylenebisacetamide (HMBA). The first two classes of hybrids were between an RA- line (also unresponsive to HMBA) that lacks cellular RA binding protein (cRABP) activity and HMBA- lines which possess cRABP and differentiate in the presence of RA. All of the hybrid clones possessed cRABP and differentiated normally upon exposure to either RA or HMBA. When the aforementioned RA- mutant was fused with a second mutant which was refractory to RA and HMBA but possessed cRABP activity, the resultant hybrid clones were responsive to both RA and HMBA and had cRABP activity. These results suggest that all of these mutants were recessive and complementary. Tumors from these hybrid lines differentiated extensively, in some instances much more so than the mutant parental lines and even the wild-type lines from which the mutants were derived. Based upon these observations, we propose that various EC lines might differentiate poorly in tumor form for different reasons. Hybrids between two differentiation-defective, cRABP- lines appeared to be at least partially complemented for responsiveness to RA and HMBA. These hybrids contained low but detectable levels of cRABP. This is not a consequence of tetraploidy since fusions between cells from the same mutant line retained their differentiation-defective phenotype and possessed little or no cRABP activity. Unlike tumors from the other hybrids described above, tumors from these hybrid lines expressed a very restricted pattern of differentiated cell types. This might be because the mutant lines in the latter hybrids originally derived from the same wild-type EC line.     

Mutant alleles for hypoxanthine phosphoriboxyltransferase: 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 phosphoribosyltransferase (HPRT) in three such mutants have been found to exhibit an increased Km for 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.     

Expression of recessive Aprt- mutations in mouse CAK cells resulting from chromosome loss and duplication

Karyotypes of recessive mutants at the autosomal adenine phosphoribosyltransferase (Aprt) locus in a clone of the near-diploid mouse CAK cell line have been analyzed. The Aprt located on chromosome 8. One copy of chromosome 8 was morphologically abnormal in the parental clone (CAK-B3-Toyr13) from which Aprt- mutants were isolated. Among 22 mutants, there were ten in which one copy of chromosome 8 had been lost. Four of these were monosomic, and in the others duplication of the remaining homolog had occurred. These findings indicate that newly induced recessive mutations in cultured mammalian cells can be expressed as the result of loss of one chromosome carrying a wild-type allele with or without duplication of the homolog carrying the mutant allele. Loss and duplication would not be detected in cell lines lacking morphologically marked chromosomes.     

Anchoring and degradation of glycolipid-anchored membrane proteins by L929 versus by LM-TK- mouse fibroblasts: implications for anchor biosynthesis.

Although many cells anchor surface proteins via moieties that are sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC), the anchor moieties of surface proteins of mouse L929 cells resist PI-PLC. By constructing stable hybrids between L929 and lymphoma cells that express glycolipid-anchored proteins in a PI-PLC-sensitive form, we show that PI-PLC resistance behaves as a recessive trait. Since putative mannolipid precursors of the lipid anchors bear alkali-labile substituents which make them resist PI-PLC, these observations are most simply interpreted by postulating that L929 lacks a critical anchor deacylase. Unlike the L929 cell line, two of its descendants, the LM cell line and its thymidine kinase-negative variant (LM-TK-), do not express glycolipid-anchored proteins on their surface. Moreover, unlike L929 cells, LM-TK- cells rapidly inactivate at least one lipid-anchored enzyme in a compartment sensitive to acidotropic amines and leupeptin. By fusion of LM-TK- cells to mouse Thy-1- lymphoma mutants and monitoring of surface expression of lipid-anchored proteins, we assign LM-TK- to lymphoma mutant complementation group H. This genetic assignment is matched by analysis of mannolipids of L929, LM-TK-, wild-type, and class H lymphoma mutant cells: striking similarities are seen between the two wild-type cells by contrast to the mutants. Since the differences pertain to lipids which have properties consistent with their being anchor precursors, we suggest that LM-TK- has a lesion in the synthesis of anchor precursor mannolipids.