Mechanism of conjugation and recombination in bacteria XVI

Summary Protein synthesis by ribosomes from several cryptopleurine-resistant yeast mutants is also resistant to emetine and tubulosine. These mutants can be classified into two different types: Class I mutants which display high levels of resistance to emetine and tubulosine and Class II mutants that are only weakly resistant to tubulosine and are slightly more sensitive to emetine than those of Class I. Apparently all mutants have similar levels of resistance to cryptopleurine. The distinct phenotypes of Class I and Class II strains are expressed through their 40S ribosomal subunit. Genetic analysis has shown that the mutations to cryptopleurine resistance are allelic and that in a particular case (strain CRY6) the pleiotropic phenotype is a result of the expression of the cryl locus. It is suggested that Class I and Class II mutants arise from two independent mutational events within the cryl allele. in heterozygous (+/cryl) diploids both the sensitive and the resistant genes are expressed as shown by studies of the action of cryptopleurine on polyphenylalanine-synthesizing system derived from each parental sensitive and resistant haploid strain and heterozygous diploid strains. The apparent dominance of sensitivity over resistance which may be observed in vivo in heterozygous (+/cryl) diploids has been explained in terms of the mode of action of the inhibitors.     

An in vitro analysis of the dominance of emetine sensitivity in Chinese hamster ovary cell hybrids

The behavior of ribosomes derived from EmtR X EmtS hybrid cells in in vitro protein synthesis is similar to that observed with a 1:1 mixture of ribosomes from EmtR and EmtS cells. When mRNA (BM virus RNA) is present in limiting amounts (RNA/ribosome molar ratio = 0.1), protein synthesis in either mixture is sensitive to emetine. In contrast, when mRNA is present in excess (RNA/ribosome molar ratio = 2), the emetine resistant as well as the sensitive components are both expressed in the mixtures. These results strongly indicate that emetine resistant and sensitive ribosomes are present in the hybrid cells in about equal amounts and that the dominance of emetine sensitivity is best explained by assuming that emetine acts by blocking ribosome movement along mRNA by inhibiting the translocation step. The observed time lag in the expression of EmtRI and EmtRII mutations following mutagenesis is consistent with the above hypothesis for the mechanism of action of emetine.     

Structure-activity relationships between cryptopleurine and related compounds acting on yeast cell-free systems

The inhibitory effects on poly(U)-directed polyphenylalanine synthesis of cryptopleurine and a number of structurally related analogs have been compared in a yeast cell-free system. Results suggest that the quinolidine structure by itself does not promote biological activity, and for an inhibitory effect it must be condensed with a phenanthrene or another related compound such as naphthalene. The results are presented and possible relationships between structure and activity for the compounds emetine, tubolosine, tylophora alkaloids, and various cryptopleurine analogs are considered.     

Genetic and biochemical distinction among Chinese hamster cell emtA, emtB, and emtC mutants.

Genetic and biochemical experiments have enabled us to more clearly distinguish three genetic loci, emtA, emtB, and emtC, all of which can be altered to give rise to resistance to the protein synthesis inhibitor, emetine, in cultured Chinese hamster cells. Genetic experiments have demonstrated that, unlike the emtB locus, neither the emtA locus nor the emtC locus is linked to chromosome 2 in Chinese hamster cells, clearly distinguishing the latter two genes from emtB. emtA mutants can also be distinguished, biochemically, from emtB and emtC mutants based upon different degrees of cross-resistance to another inhibitor of protein synthesis, cryptopleurine. Two-dimensional gel electrophoretic analysis of ribosomal proteins failed to detect any electrophoretic alterations in ribosomal proteins from emtA or emtC mutants that could be correlated with emetine resistance. However, a distinct electrophoretic alteration in ribosomal protein S14 was observed in an emtB mutant. In addition, the parental Chinese hamster peritoneal cell line of an emtC mutant, and the emtC mutant itself, are apparently heterozygous for an electrophoretic alteration in ribosomal protein L9.     

Modification of Ribosomes in Cryptopleurine-Resistant Mutants of Yeast

Cryptopleurine-resistant mutants of Saccharomyces cerevisiae were isolated. A single, recessive nuclear gene, very closely linked to the mating locus (2.1 centimorgans), is responsible for resistance. Ribosomes from the mutants were found to be resistant to cryptopleurine when analyzed by poly(U)-directed polyphenylalanine synthesis. Analysis of the distribution of ribosomes between monosomes and polysomes in sensitive cells exposed to cryptopleurine suggests that some step is inhibited during the elongation phase of protein synthesis.     

Pleiotropic phenotype of colchicine-resistant CHO cells: cross-resistance and collateral sensitivity.

Colchicine resistant (CHR) mutants of CHO cells with reduced permeability to colchicine display extensive cross-resistance to a number of apparently unrelated compounds including puromycin, daunomycin, emetine, ethidium bromide and gramicidin D. A positive correlation was observed between the level of cross-resistance and the relative hydrophobicity of these compounds. The mutants also showed increased (collateral) sensitivity to local anaesthetics (procaine, tetracaine, xylocaine and propanolol), steroid hormones (1-dehydrotestosterone, corticosterone and 5beta-pregnan-3,20-dione) and some Triton X compounds. In general, the degree of the pleiotropic response (cross-resistance or collateral sensitivity) correlated with the degree of colchicine resistance in mutant lines. These results are consistent with the pleiotropic phenotype being the result of the same mutation(s) which confer colchicine resistance and support a model for resistance in which the reduced permeability is assumed to be the result of an alteration in the modulation of the fluidity of the surface membrane.     

Pleiotropic changes in cycloheximide-resistant insect cell clones

Summary Somatic cell mutants resistant to drugs that interact with the eukaryotic ribosome provide a useful tool for studies on ribosome structure, function, and genetics. FromAedes albopictus (mosquito) cells, cycloheximide-resistant mutants (Cx-705 and Cx-738) that were about 30-fold more resistant to cycloheximide than the parental cells have been obtained. The observation that protein synthesis in cell-free lysates from Cx-705 and Cx-738 cells was resistant to cycloheximide led us to suspect that the alteration in these mutants might affect the ribosome. The present studies show that the cycloheximide-resistant cells grow poorly and eventually die at 34.5°C, a temperature at which wild-type cells grow normally. Relative to control cells, the cycloheximide-resistant cells show there were no differences between cycloheximide-resistant cells and wild-type cells in sensitivity to puromycin, emetine, or cryptopleurine. Cx-705 cells were predominantly diploid; in contrast, the frequency of tetraploid nuclei in Cx-738 cells was about 40%. This investigation was supported by grant AI20385 from the National Institutes of Health, Bethesda, MD and by a Basil O’Connor Starter Research Grant (5–415) from the March of Dimes Birth Defects Foundation.     

The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation.

We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.     

Expression of cryptopleurine resistance in Saccharomyces cerevisiae.

An examination of gene expression in diploids may not always be sufficient for determination of the dominant or recessive character of an allele. In Saccharomyces cerevisiae resistance to cryptopleurine has been attributed to a single recessive nuclear gene, cryl, located on chromosome III. We found, contrary to expectations, that resistance to cryptopleurine is not expressed in diploids that are monosomic for chromosome III. Examination of strains of different ploidy on gradient plates shows that the presence of the sensitive allele in a cell does not affect the level of resistance, but rather the level of resistance is directly related to the ratio of resistant alleles to the number of chromosome sets.     

Detection of inhibitors of protein and nucleic acid synthesis using oocytes of Xenopus laevis microinjected with the herpes thymidine kinase gene

We have developed an assay that measures the inhibition of protein synthesis and can be used in conjunction with a whole embryo bioassay that detects the ability of a chemical to cause fetotoxicity, malformation and abnormal growth. The assay involves microinjecting the herpes thymidine kinase gene into stage 6 oocytes of Xenopus laevis then exposing the oocytes to a test compound for 18-24 h. The inhibition of thymidine kinase (TK) expression caused by an inhibitor is then measured by simple enzyme assay. Protein synthesis inhibitors such as cycloheximide, puromycin and emetine all inhibited TK synthesis. Concentrations of cycloheximide (1.4 X 10(-4) mg/ml) and puromycin (0.04 mg/ml) near the 96 h embryo LC50 inhibited thymidine kinase expression by 78% and 97%, respectively but emetine (0.01 mg/ml) had no effect. However, 0.1 mg/ml emetine inhibited TK synthesis by almost 50%. The RNA synthesis inhibitor, actinomycin D (0.013 mg/ml) inhibited TK expression by 61%. DNA synthesis inhibitors hydroxyurea (2.0 mg/ml), cytosine arabinoside (2.0 mg/ml) and ethidium bromide (0.02 mg/ml) failed to inhibit the expression of the TK gene even though these concentrations were near the 96 h embryo LC50. The whole embryo bioassay cannot differentiate the DNA synthesis inhibitors from the RNA and protein synthesis inhibitors but the oocyte assay can. This type of molecular test data can help separate classes of teratogens such as DNA synthesis inhibitors from nonteratogenic compounds such as protein synthesis inhibitors and allow the extrapolation of test data to other species.     

Taxol resistant mutants of Chinese hamster ovary cells: genetic biochemical, and cross-resistance studies

The effects of the microtubule inhibitor taxol on the growth and viability of Chinese hamster ovary (CHO) cells have been examined. Stable mutants which are between seven to 11-fold more resistant to taxol have been selected in a single step from ethyl methanesulfonate-mutagenized CHO cells. The two taxol-resistant mutants (Tax^R-1 and Tax^R-2) which have been studied in detail exhibit novel and strikingly different cross-resistance/collateral sensitivity patterns to various microtubule inhibitors. For example, the Tax^R-1 mutant exhibits increased resistance to vinblastine, but in comparison to the parental cells, it shows enhanced sensitivity toward colchicine, colcemid, stegnacine, and griseofulvin. However, the sensitivity of this mutant toward other unrelated compounds, e.g., puromycin, daunomycin, etc., remained largely unaltered. The specific pattern of cross-resistance/collateral-sensitivity of this mutant toward various microtubule inhibitors suggests that the genetic lesion in this mutant may be affecting a microtubule-related component. The Tax^R-2 mutant, in contrast, is highly resistant to various microtubule inhibitors including colchicine, colcemid, stegnacine, maytan-sine, vinblastine, and podophyllotoxin. This mutant also exhibits greatly increased cross-resistance to daunomycin, puromycin, ethidium bromide, and VM-26 (compounds which do not inhibit microtubule assembly), and shows reduced cellular uptake of ³H-daunomycin indicating that the genetic lesion in this mutant nonspecifically affects the membrane permeability of various drugs. The cell hybrids formed between Tax^R-1 (or Tax^R-2 mutant(s)) and a taxol-sensitive cell line exhibit intermediate levels of resistance to the drug, indicating that the Tax^R phenotypes of both these mutants behave codominantly under these conditions.     

Binding of amphipathic drugs and probes to biological membranes

Bouvardain is an antitumor drug that inhibits protein synthesis in intact eukaryotic cells and cell-free systems. Our present studies have shown that bouvardin acts at the level of the 80 S ribosome in a site somehow involved with the interaction of EF1 and EF2. Indeed bouvardin inhibits EF1-dependent binding of aminoacyl-tRNA and EF2-dependent translocation of peptidyl-tRNA but does not affect the non-enzymic translocation since this relation does not require EF2. The site of the 80 S ribosome involved in the interaction with bouvardin appears to be independent from the cycloheximide and the cryptopleurine binding sites since yeast mutants resistant to cycloheximide or cryptopleurine are sensitive to bouvardin.     

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.     

Construction and characterization of Chinese hamster cell EmtA EmtB double mutants.

Starting with hybrid cell lines between a Chinese hamster cell EmtA mutant and a Chinese hamster cell EmtB mutant, we have constructed cell lines that are homozygous for mutant alleles at both the emtA locus and the emtB locus, by using a two-step segregation protocol. The EmtA EmtB double mutants are approximately 10-fold more resistant to emetine inhibition than either of the parental mutants. Having both the EmtA mutation and the EmtB mutation expressed in the same cell also results in a level of resistance to cryptopleurine that is significantly higher than a simple additive effect of the two mutations alone. Analysis of ribosomal proteins by two-dimensional polyacrylamide gel electrophoresis demonstrated that a parental hybrid and a first-step segregant, which has lost the wild-type emtA allele, synthesize both a normal and an altered form of ribosomal protein S14, whereas an EmtA EmtB double mutant synthesizes only the altered form of this ribosomal protein. This result confirms that the emtB locus is the structural gene for ribosomal protein S14. Our results also suggest that the products of the emtA and emtB loci interact directly, indicating that the emtA locus, like the emtB locus, encodes a component of the ribosome.     

Induction of hepatic tyrosine aminotransferase mRNA by protein synthesis inhibitors.

Several protein synthesis inhibitors were as effective as the inducers hydrocortisone or cyclic AMP in elevating rat liver tyrosine aminotransferase mRNA levels when assayed in the wheat germ cell-free translational system. Cycloheximide, emetine, or puromycin increased this mRNA activity 6- to 7-fold within 4 h after in vivo administration. No increase in total hepatic mRNA levels or tryptophan oxygenase mRNA was found after treatment with these protein synthesis inhibitors. Furthermesults suggest that a short lived protein may specifically regulate the level of functional hepatic tyrosine aminotransferase mRNA or that ongoing translation of this mRNA is required for its degradation.     

Cross-resistance and biochemical studies with two classes of HeLa cell mutants resistant to cardiac glycosides. The unusual behavior of cardenolide SC4453

In HeLa cells two different types of mutants resistant to the cardiac glycoside ouabain (OuaR mutants) or erythrophleum alkaloid cassaine (CasR mutants) have been obtained. One type of mutants resistant to these compounds (designated as group A) are highly resistant (between 50 and 2000-fold) to various cardiac glycosides and their genins such as ouabain, oleandrin, digitoxin, digitoxigenin, strophanthidin, convallatoxin, gitoxin, gitoxigenin, gitaloxin, bufalin, and digoxigenin, but exhibit no cross-resistance to SC4453, a digoxin analog which contains a pyridazine ring in place of the lactone ring in the C-17 position. The second type of mutants (group B) exhibit cross-resistance to all of the cardiac glycosides including SC4453, but their level of resistance is at least 5-10-fold less than that of group A mutants. Interestingly, both groups of mutants exhibited similar degree of cross-resistance towards digoxin and actodigin (AY22241), indicating some differences in their behavior from other cardiac glycosides. Both classes of mutants exhibit no cross-resistance to a wide variety of other structurally and functionally related compounds, e.g. sanguinarine nitrate, ethacrynic acid, penicillic acid, veratridine, harmaline hydrochloride, 5,5'-diphenylhydantoin, quindonium bromide, methyl quinolizinum bromide, estradiol 17 beta-acetate, 21-acetoxy-pregnenolone, vanadium pentoxide, digitonin, and adriamycin, indicating that the genetic lesions in both groups of mutants are specific for cardiac glycosides. This inference is supported by the observation that both group A and B mutants show reduced binding of [3H]ouabain. In group A mutants, a part of the Na+/K+-ATPase activity is highly resistant to inhibition by ouabain, indicating that the genetic lesion in these mutants directly affects Na+/K+-ATPase. In contrast, the Na+/K+-ATPase from the group B mutants showed similar resistance towards ouabain and SC4453 as observed for the parental HeLa cells, indicating that these mutants are affected in a cellular component, other than Na+/K+-ATPase, which is involved in the interaction of cardiac glycosides with the cells. The lack of cross-resistance of the group A mutants to SC4453 and normal sensitivity of their Na+/K+-ATPase to this compound provides strong evidence that the mechanism of interaction of SC4453 with Na+/K+-ATPase differs from that of other cardiac glycosides.     

5-Chloroindoloyl glycine amide inhibitors of glycogen phosphorylase: synthesis, in vitro, in vivo, and X-ray crystallographic characterization

The synthesis, in vitro, and in vivo biological characterization of a series of achiral 5-chloroindoloyl glycine amide inhibitors of human liver glycogen phosphorylase A are described. Improved potency over previously reported compounds in cellular and in vivo assays was observed. The allosteric binding site of these compounds was shown by X-ray crystallography to be the same as that reported previously for 5-chloroindoloyl norstatine amides.     

Induction and Characterization of Penicillium caseicolum Mutants Resistant to Ergosterol Biosynthesis Inhibitors

The isolation of Penicillium caseicolum mutants resistant to different fungicides which inhibit ergosterol biosynthesis is reported. Mutational frequencies for resistance were high (3 × 10⁻³ to 3 × 10⁻⁵). The levels of resistance toward the inhibitors of sterol C-14 demethylation were always low (<10), whereas high values were obtained with mutants resistant to inhibitors of sterol Δ14 reduction or Δ8→Δ7 isomerization, or both. Generally, there was a positive cross-resistance between fungicides showing the same biochemical mode of action but not between compounds of two different groups. Mycelial growth rate and sporulation were tested; several mutants were not affected for these characteristics. We conclude that resistance to ergosterol biosynthesis inhibitors may be used as a good marker for genetic studies through protoplast fusion.     

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.     

An integrated platform of genomic assays reveals small-molecule bioactivities

Bioactive compounds are widely used to modulate protein function and can serve as important leads for drug development. Identifying the in vivo targets of these compounds remains a challenge. Using yeast, we integrated three genome-wide gene-dosage assays to measure the effect of small molecules in vivo. A single TAG microarray was used to resolve the fitness of strains derived from pools of (i) homozygous deletion mutants, (ii) heterozygous deletion mutants and (iii) genomic library transformants. We demonstrated, with eight diverse reference compounds, that integration of these three chemogenomic profiles improves the sensitivity and specificity of small-molecule target identification. We further dissected the mechanism of action of two protein phosphatase inhibitors and in the process developed a framework for the rational design of multidrug combinations to sensitize cells with specific genotypes more effectively. Finally, we applied this platform to 188 novel synthetic chemical compounds and identified both potential targets and structure-activity relationships.