The effects of selenium and the GPx-1 selenoprotein on the phosphorylation of H2AX.

BACKGROUND: Significant data supports the health benefits of selenium although supplementation trials have yielded mixed results. GPx-1, whose levels are responsive to selenium availability, is implicated in cancer etiology by human genetic data. Selenium's ability to alter the phosphorylation of the H2AX, a histone protein that functions in the reduction of DNA damage by recruiting repair proteins to the damage site, following exposure to ionizing radiation and bleomycin was investigated. METHODS: Human cell lines that were either exposed to selenium or were transfected with a GPx-1 expression construct were exposed to ionizing radiation or bleomycin. Phosphorylation of histone H2AX was quantified by flow cytometry and survival by the MTT assay. Phosphorylation of the Chk1 and Chk2 checkpoint proteins was quantified by western blotting. RESULTS: In colon-derived cells, selenium increases GPx-1 and attenuated H2AX phosphorylation following genotoxic exposures while the viability of these cells was unaffected. MCF-7 cells and transfectants that express high GPx-1 levels were exposed to ionizing radiation and bleomycin, and H2AX phosphorylation and cell viability were assessed. GPx-1 increased H2AX phosphorylation and viability following the induction of DNA damage while enhancing the levels of activated Chk1 and Chk2. CONCLUSIONS: Exposure of mammalian cells to selenium can alter the DNA damage response and do so by mechanisms that are dependent and independent of its effect on GPx-1. GENERAL SIGNIFICANCE: Selenium and GPx-1 may stimulate the repair of genotoxic DNA damage and this may account for some of the benefits attributed to selenium intake and elevated GPx-1 activity.

Glutathione peroxidase and viral replication: implications for viral evolution and chemoprevention.

It is likely that several of the biological effects of selenium are due to its effects on selenoprotein activity. While the effects of the anti-oxidant selenoprotein glutathione peroxidase (GPx) on inhibiting HIV activation have been well documented, it is clear that increased expression of this enzyme can stimulate the replication and subsequent appearance of cytopathic effects associated with an acutely spreading HIV infection. The effects of GPx on both phases of the viral life cycle are likely mediated via its influence on signaling molecules that use reactive oxygen species, and similar influences on signaling pathways may account for some of the anti-cancer effects of selenium. Similarly, selenium can alter mutagenesis rates in both viral genomes and the DNA of mammalian cells exposed to carcinogens. Comparisons between the effects of selenium and selenoproteins on viral infections and carcinogenesis may yield new insights into the mechanisms of action of this element.

Selective inhibition of selenocysteine tRNA maturation and selenoprotein synthesis in transgenic mice expressing isopentenyladenosine-deficient selenocysteine tRNA.

Selenocysteine (Sec) tRNA (tRNA([Ser]Sec)) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA([Ser]Sec) lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA([Ser]Sec) genes into the mouse genome. Overexpression of wild-type tRNA([Ser]Sec) did not affect selenoprotein synthesis. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i(6)A(-)) tRNA([Ser]Sec) in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA([Ser]Sec) population showed that expression of i(6)A(-) tRNA([Ser]Sec) altered the distribution of the two major isoforms, whereby the maturation of tRNA([Ser]Sec) by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i(6)A(-) tRNA([Ser]Sec) and wild-type tRNA([Ser]Sec) are regulated independently and that the amount of wild-type tRNA([Ser]Sec) is determined, at least in part, by a feedback mechanism governed by the level of the tRNA([Ser]Sec) population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i(6)A(-) tRNA([Ser]Sec) transgenic mice will be useful in assessing the biological roles of selenoproteins.

Radiosensitivity of mammalian cell lines engineered to overexpress cytosolic glutathione peroxidase.

Reactive oxygen species are believed to be involved in radiation lethality. Glutathione peroxidase is an intracellular enzyme with antioxidant functions. To determine whether increasing the cellular antioxidant capacity can confer radiation resistance, the effect of overexpression of glutathione peroxidase on radiosensitivity was determined in two different cell types. An expression construct including the bovine cytosolic glutathione peroxidase cDNA was used to overexpress this enzyme in cells of the human lymphoblast cell line Sup-T1 as well as the Chinese hamster ovary cell line AA8. Supplementation of the culture media with 30 nM sodium selenite was included to obtain optimal glutathione peroxidase activity. Northern blot analysis confirmed the presence of the construct mRNA, and a standard coupled spectrophotometric assay demonstrated significantly increased glutathione peroxidase activity in the transfected cell lines. An approximately 8-fold increase was found in the Sup-T1 cells, and an approximately 30-fold increase was obtained in the Chinese hamster ovary AA8 cells. Clonogenic survival was assayed in the overexpressing cells and compared to that in control cells transfected with vector alone. Despite significantly increased glutathione peroxidase activity, no observable radioprotection was conferred in either of the two cell lines studied, indicating that increased glutathione peroxidase activity is insufficient to confer radioresistance in the two cell types examined. These data are discussed in the context of using antioxidants as adjuncts to clinical radiotherapy.

Distribution and functional consequences of nucleotide polymorphisms in the 3'-untranslated region of the human Sep15 gene.

Selenium has been shown to prevent cancer in a variety of animal model systems. Both epidemiological studies and supplementation trials have supported its efficacy in humans. However, the mechanism by which selenium suppresses tumor development remains unknown. Selenium is present in known human selenoproteins as the amino acid selenocysteine (Sec). Sec is inserted cotranslationally in response to UGA codons within selenoprotein mRNAs in a process requiring a sequence within the 3'-untranslated region (UTR), referred to as a Sec insertion sequence (SECIS) element. Recently, a human Mr 15,000 selenoprotein (Sep15) was identified that contains an in-frame UGA codon and a SECIS element in the 3'-UTR. Examination of the available cDNA sequences for this protein revealed two polymorphisms located at position 811 (C/T) and at position 1125 (G/A) located within the 3'-UTR. Here, we demonstrate significant differences in Sep15 allele frequencies by ethnicity and that the identity of the nucleotides at the polymorphic sites influences SECIS function in a selenium-dependent manner. This, together with genetic data indicating loss of heterozygosity at the Sep15 locus in certain human tumor types, suggests that Sep15 may be involved in cancer development, risk, or both.

Structure-expression relationships of the 15-kDa selenoprotein gene. Possible role of the protein in cancer etiology.

Selenium has been implicated in cancer prevention, but the mechanism and possible involvement of selenoproteins in this process are not understood. To elucidate whether the 15-kDa selenoprotein may play a role in cancer etiology, the complete sequence of the human 15-kDa protein gene was determined, and various characteristics associated with expression of the protein were examined in normal and malignant cells and tissues. The 51-kilobase pair gene for the 15-kDa selenoprotein consisted of five exons and four introns and was localized on chromosome 1p31, a genetic locus commonly mutated or deleted in human cancers. Two stem-loop structures resembling selenocysteine insertion sequence elements were identified in the 3'-untranslated region of the gene, and only one of these was functional. Two alleles in the human 15-kDa protein gene were identified that differed by two single nucleotide polymorphic sites that occurred within the selenocysteine insertion sequence-like structures. These 3'-untranslated region polymorphisms resulted in changes in selenocysteine incorporation into protein and responded differently to selenium supplementation. Human and mouse 15-kDa selenoprotein genes manifested the highest level of expression in prostate, liver, kidney, testis, and brain, and the level of the selenoprotein was reduced substantially in a malignant prostate cell line and in hepatocarcinoma. The expression pattern of the 15-kDa protein in normal and malignant tissues, the occurrence of polymorphisms associated with protein expression, the role of selenium in differential regulation of polymorphisms, and the chromosomal location of the gene may be relevant to a role of this protein in cancer.

Multiple levels of regulation of selenoprotein biosynthesis revealed from the analysis of human glioma cell lines.

To gain a better understanding of the biological consequences of the exposure of tumor cells to selenium, we evaluated the selenium-dependent responses of two selenoproteins (glutathione peroxidase and the recently characterized 15-kDa selenoprotein) in three human glioma cell lines. Protein levels, mRNA levels, and the relative distribution of the two selenocysteine tRNA isoacceptors (designated mcm(5)U and mcm(5)Um) were determined for standard as well as selenium-supplemented conditions. The human malignant glioma cell lines D54, U251, and U87 were maintained in normal or selenium-supplemented (30 nM sodium selenite) conditions. Northern blot analysis demonstrated only minor increases in steady-state GSHPx-1 mRNA in response to selenium addition. Baseline glutathione peroxidase activity was 10.7 +/- 0.7, 7.6 +/- 0.7, and 4.3 +/- 0.7 nmol NADPH oxidized/min/mg protein for D54, U251, and U87, respectively, as determined by the standard coupled spectrophotometric assay. Glutathione peroxidase activity increased in a cell line-specific manner to 19.7 +/- 1.4, 15.6 +/- 2.1, and 6. 7 +/- 0.5 nmol NADPH oxidized/min/mg protein, respectively, as did a proportional increase in cellular resistance to H(2)O(2), in response to added selenium. The 15-kDa selenoprotein mRNA levels likewise remained constant despite selenium supplementation. The selenium-dependent change in distribution between the two selenocysteine tRNA isoacceptors also occurred in a cell line-specific manner. The percentage of the methylated isoacceptor, mcm(5)Um, changed from 35.5 to 47.2 for D54, from 38.1 to 47.3 for U251, and from 49.0 to 47.6 for U87. These data represent the first time that selenium-dependent changes in selenoprotein mRNA and protein levels, as well as selenocysteine tRNA distribution, were examined in human glioma cell lines.

Analysis of selenocysteine (Sec) tRNA([Ser]Sec) genes in Chinese hamsters.

Several recent observations have indicated that the primary structure of the Chinese hamster selenocysteine tRNA([Ser]sec) is different than those of other mammalian species. These reports prompted us to investigate the gene sequence for this tRNA in Chinese hamsters. Southern blotting of Chinese hamster ovary (CHO) genomic DNA derived from cultured cells with a tRNA([Ser]sec) probe indicated several hybridizing bands, and each of the corresponding genetic loci was isolated from a recombinant CHO library by molecular cloning. Sequence analysis of these regions indicated three likely pseudogenes and a single functional gene whose sequence differed from those of other mammals. Of these, only one pseudogene and the putative functional gene are actively transcribed following their microinjection into Xenopus oocytes. The possibility that the functional CHO tRNA([Ser]sec) evolved from an edited transcript is discussed.

Selenium metabolism in Drosophila. Characterization of the selenocysteine tRNA population.

The selenocysteine (Sec) tRNA population in Drosophila melanogaster is aminoacylated with serine, forms selenocysteyl-tRNA, and decodes UGA. The Km of Sec tRNA and serine tRNA for seryl-tRNA synthetase is 6.67 and 9.45 nM, respectively. Two major bands of Sec tRNA were resolved by gel electrophoresis. Both tRNAs were sequenced, and their primary structures were indistinguishable and colinear with that of the corresponding single copy gene. They are 90 nucleotides in length and contain three modified nucleosides, 5-methylcarboxymethyluridine, N6-isopentenyladenosine, and pseudouridine, at positions 34, 37, and 55, respectively. Neither form contains 1-methyladenosine at position 58 or 5-methylcarboxymethyl-2'-O-methyluridine, which are characteristically found in Sec tRNA of higher animals. We conclude that the primary structures of the two bands of Sec tRNA resolved by electrophoresis are indistinguishable by the techniques employed and that Sec tRNAs in Drosophila may exist in different conformational forms. The Sec tRNA gene maps to a single locus on chromosome 2 at position 47E or F. To our knowledge, Drosophila is the lowest eukaryote in which the Sec tRNA population has been characterized to date.

Overproduction of selenocysteine tRNA in Chinese hamster ovary cells following transfection of the mouse tRNA[Ser]Sec gene.

Selenocysteine insertion during selenoprotein biosynthesis begins with the aminoacylation of selenocysteine tRNA[ser]sec with serine, the conversion of the serine moiety to selenocysteine, and the recognition of specific UGA codons within the mRNA. Selenocysteine tRNA[ser]sec exists as two major forms, differing by methylation of the ribose portion of the nucleotide at the wobble position of the anticodon. The levels and relative distribution of these two forms of the tRNA are influenced by selenium in mammalian cells and tissues. We have generated Chinese hamster ovary cells that exhibit increased levels of tRNA[ser]sec following transfection of the mouse tRNA[ser]sec gene. The levels of selenocysteine tRNA[ser]sec in transfectants increased proportionally to the number of stably integrated copies of the tRNA[ser]sec gene. Although we were able to generate transfectants overproducing tRNA[ser]sec by as much as tenfold, the additional tRNA was principally retained in the unmethylated form. Selenium supplementation could not significantly affect the relative distributions of the two major selenocysteine tRNA[ser]sec isoacceptors. In addition, increased levels of tRNA[ser]sec did not result in measurable alterations in the levels of selenoproteins, including glutathione peroxidase.

Effects of 1,2-naphthoquinones on human tumor cell growth and lack of cross-resistance with other anticancer agents.

The sensitivity of human tumor and rat prostate tumor cells to a series of naphthoquinones, including tricyclic compounds of the beta-lapachone and dunnione families as well as 4-alkoxy-1,2-naphthoquinones, was evaluated. To better understand the mechanism of cytotoxicity of 1,2-naphthoquinones, the roles of various resistance mechanisms including P-glycoprotein, multidrug resistant associated protein, glutathione (GSH) and related enzymes, altered topoisomerase activity, and overexpression of genes that control apoptosis (bcl-2 and bc-xL) were studied. MCF7 cells were most sensitive to the naphthoquinones with IC50 values ranging from 1.1 to 10.8 microM, as compared to 2.5 to >32 microM for HT29 human colon, A549 human lung, CEM leukemia and AT3.1 rat prostate cancer cells. MCF7 ADR cells, selected for resistance to adriamycin (ADR), displayed cross-resistance to the tricyclic 1,2-naphthoquinones. Drug efflux via a P-glycoprotein mechanism was ruled out as a mechanism of resistance to 1,2-naphthoquinones, since KB-V1 cells expressing high levels of P-glycoprotein and the KB-3.1 parent line were equally sensitive to these compounds. Any resistance of the tricyclic naphthoquinones noted in ADR-resistant cells appeared to relate to the GSH redox cycle and could be circumvented by exposure to buthionine sulfoximine or by changing the structure from a tricyclic derivative to a 4-alkoxy-1,2-naphthoquinone. The 1,2-naphthoquinones were found to be cytotoxic against CEM/VM-1 and CEM/M70-B1 cells that were selected for resistance to teniposide or merbarone, respectively. In addition, cells overexpressing bcl-2 or bcl-xL proteins were as sensitive to 1,2-naphthoquinones as were control cells. Because of their effectiveness in drug-resistant cells, these agents appear to hold promise as effective chemotherapeutic agents.

Antioxidant defenses influence HIV-1 replication and associated cytopathic effects.

HIV-infected cells often exhibit reduced levels of antioxidant enzymes and thiols. To investigate the role of cellular antioxidant defenses in the progression of an acutely spreading HIV-1 infection, human Sup-T1 T cells were engineered to overexpress the selenium-dependent glutathione peroxidase, GSHPx-1. This enzyme represents a major cellular defense mechanism against toxicity associated with reactive oxygen species (ROS). T cells engineered to produce elevated GSHPx-1 activity displayed accelerated viral replication and associated cytopathic effects compared to control cells. Conversely, the inhibition of the synthesis of glutathione with buthione sulfoximine (BSO) resulted in the attenuation of viral replication in Sup-T1 cells. Similarly, exposure of human peripheral blood lymphocytes (PBLs) to low, nontoxic levels of BSO resulted in an approximately 80% decline in HIV-1 replication as indicated by Western blot analysis of viral proteins.

A T-cell model for the biological role of selenium-dependent glutathione peroxidase.

The cytosolic form of selenium-dependent glutathione peroxidase detoxifies both hydrogen and lipid peroxides and therefore represents a major component of the cellular anti-oxidant defenses. In order to study the biological role of this enzyme, we generated an expression construct in a retroviral vector, which when introduced into immortalized human T-cells, resulted in significant increases in the activity of this important enzyme. This effect is stable over extended maintenance in culture. The anti-oxidant defenses in these same cells are also shown to be attenuated by chemically reducing cellular glutathione levels. Collectively, the ability to both increase and decrease the anti-oxidant defenses in human T cells results in a useful model system for the study of oxidative stress and signaling in this cell type.

Selenocysteine tRNA[Ser]Sec levels and selenium-dependent glutathione peroxidase activity in mouse embryonic stem cells heterozygous for a targeted mutation in the tRNA[Ser]Sec gene.

To investigate the effect of a reduced level of selenocysteine (Sec) tRNA[Ser]Sec in selenoprotein biosynthesis, two mouse embryonic stem (ES) cell lines heterozygous for the corresponding gene were generated by homologous recombination of the host genome with targeting vectors encoding a deleted or a disrupted tRNA[Ser]Sec gene. The presence of a single functional gene in ES cells afforded us an opportunity to determine directly in the cell line the effect of reduced gene dosage on (1) the levels of the Sec tRNA[Ser]Sec population, (2) the distributions of the isoacceptors within the Sec tRNA population, and (3) selenoprotein biosynthesis. We therefore determined the amounts and distributions of the two major tRNA[Ser]Sec isoacceptors, designated mcm5U and mcm5Um, within the Sec tRNA population and determined the activity of the anti-oxidant, selenium-containing glutathione peroxidase (GPx) in the heterozygotes and in wild type cells grown in media with and without added selenium. The level of the Sec tRNA[Ser]Sec population in the heterozygotes was approximately 60% of that of wild type cells grown in media under normal conditions, while the ratio of the mcmU isoacceptor in wild type vs mutant cells was approximately 2:1 and of the mcmUm isoacceptor approximately 1:1. In the presence of media supplemented with selenium, the Sec tRNA[Ser]Sec population increased about 20% in wild type cells and virtually not all in heterozygous cells, and the level of the Sec tRNA[Ser]Sec population was, therefore, approximately 50% of that of wild type cells. GPx activity was indistinguishable among these cell lines in either selenium-supplemented or unsupplemented media, indicating that the resultant changes in tRNA[Ser]Sec levels did not have a measurable effect on GPx biosynthesis.

The inhibition of radiation-induced mutagenesis by the combined effects of selenium and the aminothiol WR-1065.

In order to evaluate the anti-mutagenic effects of the potential chemoprotective compounds selenium and (S)-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-1065), CHO AA8 cells were exposed to both compounds either individually or in combination prior to irradiation. Mutation frequency following exposure to 8 Gy was evaluated by quantitation of the mutations detected at the hprt locus of these cells. Protection against radiation-induced mutation was observed for both 30 nM sodium selenite or 4 mM WR-1065. In addition, the protection against mutation induction provided by the combination of these agents appeared additive. In contrast, sodium selenite did not provide protection against radiation toxicity when provided either alone or in conjunction with WR-1065. In order to evaluate the possible mechanisms of the anti-mutagenic effects observed in these cells, glutathione peroxidase (GPx) activity was evaluated following exposure to the chemopreventative compounds. The addition of sodium selenite to the culture media resulted in a 5-fold increase in GPx activity, which was unaltered by the presence of the WR-1065. Northern analysis of RNA derived from these cells indicated that selenium supplementation resulted in a marginal increase in the mRNA for the cytosolic GPx (GSHPx-1) which was insufficient to account for the stimulation of GPx activity observed in cellular extracts. These results suggest that selenium and WR-1065 offer protection via independent mechanisms and that GPx stimulation remains a possible mechanism of the anti-mutagenic effect of selenium.

bc1-2 expression facilitates human immunodeficiency virus type-1 mediated cytopathic effects during acute spreading infections.

The cytopathic effects (CPE) resulting from the infection of CD4+ T cells by human immunodeficiency virus (HIV) have generally been characterized as single-cell killing associated with apoptosis and/or the generation of syncytia resulting from the direct cell-to-cell transmission of the virus. Little is known, however, about the cellular factors influencing host cell susceptibility to HIV-mediated CPE. Because expression of the antiapoptosis gene, bcl-2, enhances cell viability after exposure to cytotoxic agents or stimuli, the effect of bcl-2 expression on HIV infection of stably transfected T-cell clones was investigated. Unexpectedly, bcl-2 expression by these cells accelerated the kinetics of an acute spreading HIV infection, as evidenced by a rapid loss of culture viability associated with the appearance of CPE and reverse transcriptase activity in the culture supernatant. This unexpected effect of bcl-2 expression results from the arrest of syncytial apoptosis, directly facilitating the cell-to-cell transmission of HIV. In addition, bcl-2 expression is associated with enhanced HIV replication as determined by HIV type 1-specific Western blot (immunoblot) analysis. These results suggest that the inhibition of apoptosis is essential for this mode of viral transmission.

Lovastatin effects on human breast carcinoma cells. Differential toxicity of an adriamycin-resistant derivative and influence on selenocysteine tRNAS.

Selenocysteine tRNA[Ser]Sec isoacceptors contain the modified nucleotide i6A immediately 3' to the anticodon. Because synthesis of i6A is expected to be inhibited by lovastatin, the status of tRNA[Ser]Sec isoacceptors was examined in human breast carcinoma cells. As part of the initial characterization of these cells, it was determined that an adriamycin resistant derivative of the MCF-7 cell line exhibited a dramatic increase in the sensitivity to the killing effects of lovastatin relative to the parental MCF-7 cells. When MCF-7Adr cells were incubated with high levels of lovastatin, there was a dramatic perturbation in the distribution of isoacceptors within the selenocysteine tRNA population. Lovastatin may therefore be a useful reagent for both the study of differential killing of drug-resistant tumor cells and selenoprotein biosynthesis.

Sequence and unusual 3' flanking region of the rat tRNA[Ser]Sec gene.

The single rat selenocysteine tRNA (tRNA[Ser]Sec) locus, including flanking sequence, was isolated by molecular cloning and its nucleotide (nt) sequence determined. In addition to the identification of likely regulatory elements 5' of this gene, this analysis also revealed a novel 3' repeat element consisting of three and a half repetitions of a 34-nt unit.

Varying levels of radioprotection from the effects of JANUS neutrons in repair-deficient xrs-5 hamster cells treated with azacytidine.

A series of cell lines have been generated from the radiation-sensitive Chinese hamster ovary line xrs-5 by treatment with azacytidine. Several of these lines have been shown to be resistant to gamma radiation. Survival curves have been generated for several of these lines and the parental lines after exposure to 0 to 5 Gy of JANUS neutrons in the presence or absence of a 30-min pretreatment with the aminothiol radioprotector WR-1065. These studies were performed to determine whether the parental xrs-5 cell line was radioresistant to exposure to JANUS neutrons and whether reversion to a neutron-resistant phenotype correlated with recovery of aminothiol radioprotection. Exposure to 4 mM WR-1065 enhanced survival after exposure to neutron radiation for most "revertant" lines, although the increase in survival varied. The xrs-5 cell line was sensitive to JANUS neutrons and showed no protection by WR-1065. These data indicate that xrs-5 cells are also sensitive to neutron radiation, that azacytidine-induced revertants for gamma-ray survival demonstrate the wild-type phenotype for survival after neutron exposure, and that the gene product that is defective is responsible for repairing only a small portion of neutron-induced damage.

Reconstitution of the biosynthetic pathway of selenocysteine tRNAs in Xenopus oocytes.

Selenocysteine is cotranslationally introduced into a growing polypeptide in response to certain UGA codons in selenoprotein mRNAs. The biosynthesis of this amino acid initiates by aminoacylation of specific tRNAs (designated tRNA([Ser]Sec)) with serine and subsequent conversion of the serine moiety to selenocysteine. The resulting selenocysteyl-tRNA then donates selenocysteine to protein. In most higher vertebrate cells and tissues examined, multiple selenocysteine isoacceptors have been described. Two of these have been determined to differ by only a single modified residue in the wobble position of the anticodon. In addition, the steady-state levels and relative distributions of these isoacceptors have been shown to be influenced by the presence of selenium. In order to gain a better understanding of the relationship between these tRNAs and how they are regulated, both the Xenopus selenocysteine tRNA gene and an in vitro synthesized RNA have each been injected into Xenopus oocytes and their maturation analyzed. In this system, selenium enhanced RNA stability and altered the distribution of isoacceptors that differ by a single ribose methylation. Interestingly, the biosynthesis of one of these modified nucleosides (5-methylcarboxymethyl-2'-O-methyluridine), which has been identified only in the wobble position of selenocysteine tRNA, also occurs in oocytes. Examination of the modified residues in both the naturally occurring Xenopus selenocysteine tRNA and the products generated from exogenous templates in oocytes demonstrated the faithful reconstruction of the biosynthetic pathway for these tRNAs.