Reversal of mitomycin C resistance by overexpression of bioreductive enzymes in Chinese hamster ovary cells.

The clinical utility of antineoplastic agents is limited by the development of drug resistance by tumors. Mitomycin C (MC) is a bacterial product that must be enzymatically reduced to exert anticancer activity. We have demonstrated that expression of the bacterial MC resistance-associated (MCRA) protein in Chinese hamster ovary (CHO) cells confers profound resistance to this antibiotic under aerobic conditions, but not under hypoxia. MCRA produces resistance to MC by redox cycling of the activated hydroquinone intermediate back to the prodrug form. A CHO cell line developed by stepwise exposure to increasing concentrations of MC likewise expressed high level resistance to MC in air, but not under hypoxia. The overexpression of DT-diaphorase and NADPH:cytochrome c (P-450) reductase, two enzymes known to activate MC, restored sensitivity to MC in both MCRA-transfected and drug-selected cell lines. The level of sensitization was proportional to the quantity of enzyme activity expressed, supporting the concept that the levels of these two activating enzymes are important for sensitivity to MC. The findings of resistance to MC in air but not under hypoxic conditions and of restoration of sensitivity to MC by increasing levels of DT-diaphorase activity, properties not adequately explained by other resistance mechanisms (i.e., decreases in MC activation, repair of DNA lesions, and/or drug efflux), support the hypothesis that a functional mammalian homologue of MCRA may be involved in producing resistance to MC.

A twin study of chronic fatigue.

OBJECTIVE: The etiology of chronic fatigue syndrome is unknown, but genetic influences may be important in its expression. Our objective was to assess the role of genetic and environmental factors in unexplained chronic fatigue. METHODS: A classic twin study was conducted using 146 female-female twin pairs, of whom at least one member reported > or =6 months of fatigue. After completing questionnaires on symptoms, zygosity, physical health, and a psychiatric interview, twins were classified using three increasingly stringent definitions: 1) chronic fatigue for > or =6 months, 2) chronic fatigue not explained by exclusionary medical conditions, and 3) idiopathic chronic fatigue not explained by medical or psychiatric exclusionary criteria of the chronic fatigue syndrome case definition. Concordance rates in monozygotic and dizygotic twins were calculated for each fatigue definition along with estimates of the relative magnitude of genetic and environmental influences on chronic fatigue. RESULTS: The concordance rate was higher in monozygotic than dizygotic twins for each definition of chronic fatigue. For idiopathic chronic fatigue, the concordance rates were 55% in monozygotic and 19% in dizygotic twins (p =.042). The estimated heritability in liability was 19% (95% confidence interval = 0-56) for chronic fatigue > or =6 months, 30% (95% confidence interval = 0-81) for chronic fatigue not explained by medical conditions, and 51% (95% confidence interval = 7-96) for idiopathic chronic fatigue. CONCLUSIONS: These results provide evidence supporting the familial aggregation of fatigue and suggest that genes may play a role in the etiology of chronic fatigue syndrome.

Inhibition of DNA cross-linking by mitomycin C by peroxidase-mediated oxidation of mitomycin C hydroquinone.

Mitomycin C requires reductive activation to cross-link DNA and express anticancer activity. Reduction of mitomycin C (40 microm) by sodium borohydride (200 microm) in 20 mm Tris-HCl, 1 mm EDTA at 37 degrees C, pH 7.4, gives a 50-60% yield of the reactive intermediate mitomycin C hydroquinone. The hydroquinone decays with first order kinetics or pseudo first order kinetics with a t(12) of approximately 15 s under these conditions. The cross-linking of T7 DNA in this system followed matching kinetics, with the conversion of mitomycin C hydroquinone to leuco-aziridinomitosene appearing to be the rate-determining step. Several peroxidases were found to oxidize mitomycin C hydroquinone to mitomycin C and to block DNA cross-linking to various degrees. Concentrations of the various peroxidases that largely blocked DNA cross-linking, regenerated 10-70% mitomycin C from the reduced material. Thus, significant quantities of products other than mitomycin C were produced by the peroxidase-mediated oxidation of mitomycin C hydroquinone or products derived therefrom. Variations in the sensitivity of cells to mitomycin C have been attributed to differing levels of activating enzymes, export pumps, and DNA repair. Mitomycin C hydroquinone-oxidizing enzymes give rise to a new mechanism by which oxic/hypoxic toxicity differentials and resistance can occur.

Bioreductive metabolism of mitomycin C in EMT6 mouse mammary tumor cells: cytotoxic and non-cytotoxic pathways, leading to different types of DNA adducts. The effect of dicumarol.

The six DNA adducts formed in EMT6 mouse mammary tumor cells upon treatment with mitomycin C (MC) fall into two groups: (1) four guanine adducts of MC and (2) two guanine adducts derived from 2,7-diaminomitosene (2,7-DAM), the major reductive metabolite of MC. The two groups of adducts were proposed to originate from two pathways arising from reductive activation of MC: (a) direct alkylation of DNA and (b) formation of 2,7-DAM, which then alkylates DNA. The aim of this study was to test the validity of this proposal and to evaluate the significance of alkylation of DNA by 2,7-DAM. Treatment of the cells with 2,7-DAM itself yielded the same 2,7-DAM-guanine adducts as treatment with MC; however, 2,7-DAM was approximately 100-fold less cytotoxic than MC. The uptake and efflux of 2,7-DAM by EMT6 cells was comparable to that of MC, but 2,7-DAM alkylated DNA with higher efficiency than MC. These results validate the two proposed pathways and show that formation of 2,7-DAM-DNA adducts in MC-treated cells represents a relatively non-toxic pathway of reductive metabolism of MC. A selective stimulatory effect of dicumarol (DIC) on 2,7-DAM-DNA adduct formation in EMT6 cells treated with MC was also investigated. DIC had no effect on alkylation by MC in cell-free systems, nor did it have significant effects on adduct formation or cell survival for cells treated with 2,7-DAM. It is proposed that in the cell DIC stimulates a reductase enzyme located at subcellular sites where the activated MC species has no direct access to DNA and therefore is diverted into the non-cytotoxic pathway, which leads to the formation of 2,7-DAM and its adducts.

Comorbid clinical conditions in chronic fatigue: a co-twin control study.

OBJECTIVES: Chronically fatiguing illness, defined as fatigue for at least 6 months, has been associated with various physical health conditions. Our objective was to determine whether there is a significant relationship between chronically fatiguing illness and 10 clinical conditions that frequently appear to be associated with fatigue, adjusting for the potentially confounding effects of psychiatric illness. DESIGN: A co-twin control study controlling for genetic and many environmental factors by comparing chronically fatigued twins with their nonfatigued co-twins. SETTING: A nationally distributed volunteer twin registry. PARTICIPANTS: The study included 127 twin pairs in which one member of the pair experienced fatigue of at least 6 months' duration and the co-twin was healthy and denied chronic fatigue. Fatigued twins were classified into 3 levels using increasingly stringent diagnostic criteria. MEASUREMENTS AND MAIN RESULTS: Twins reported on a history of fibromyalgia, irritable bowel syndrome, multiple chemical sensitivities, temporomandibular disorder, interstitial cystitis, postconcussion syndrome, tension headache, chronic low back pain, chronic pelvic pain (women), and chronic nonbacterial prostatitis (men). The prevalence of these comorbid clinical conditions was significantly higher in the fatigued twins compared to their nonfatigued co-twins. Most notably, compared to their nonfatigued co-twins, the chronically fatigued twins had higher rates of fibromyalgia (> 70% vs < 10%) and irritable bowel syndrome (> 50% vs < 5%). The strongest associations were observed between chronic fatigue and fibromyalgia (odds ratios > 20), irritable bowel syndrome, chronic pelvic pain, multiple chemical sensitivities, and temporomandibular disorder (all with odds ratios > or = 4). Regression analysis suggested that the number of comorbid clinical conditions associated with chronic fatigue could not be attributed solely to psychiatric illness. CONCLUSIONS: Chronically fatiguing illnesses were associated with high rates of many other clinical conditions. Thus, patients with chronic fatigue may present a complex clinical picture that poses diagnostic and management challenges. Nonetheless, clinicians should assess such patients for the presence of comorbid clinical conditions. Future research should provide a better understanding of the temporal relationship of the onset of fatigue and these conditions, and develop strategies for early intervention.

Structure of adduct X, the last unknown of the six major DNA adducts of mitomycin C formed in EMT6 mouse mammary tumor cells.

Treatment of EMT6 mouse mammary tumor cells with mitomycin C (MC) results in the formation of six major MC-DNA adducts. We identified the last unknown of these ("adduct X") as a guanine N(2) adduct of 2, 7-diaminomitosene (2,7-DAM), in which the mitosene is linked at its C-10 position to guanine N(2). The assigned structure is based on UV and mass spectra of adduct X isolated directly from the cells, as well as on its difference UV, second-derivative UV, and circular dichroism spectra, synthesis from [8-(3)H]deoxyguanosine, and observation of its heat stability. These tests were carried out using 17 microg of synthetic material altogether. The mechanism of formation of adduct X involves reductive metabolism of MC to 2,7-DAM, which undergoes a second round of reductive activation to alkylate DNA, yielding adduct X and another 2,7-DAM-guanine adduct (adduct Y), which is linked at guanine N7 to the mitosene. Adduct Y has been described previously. Adduct X is formed preferentially at GpC, while adduct Y favors the GpG sequence. In contrast to MC-DNA adducts, the 2,7-DAM-DNA adducts are not cytotoxic.

The Chronic Fatigue Twin Registry: method of construction, composition, and zygosity assignment.

Chronic fatigue syndrome (CFS) and the symptom of chronic fatigue are conditions of unknown etiology. The Centers for Disease Control and Prevention (CDC) define CFS as an illness characterized by > or = 6 months of disabling fatigue associated with muscle pain, pharyngitis, and alterations in mood, sleep and neurocognition. We constructed a registry of twins with chronic fatigue to facilitate research on the impact of illness, the associated medical and psychosocial factors, and the heterogeneous proposed mechanisms for these conditions. We have recruited 204 twin pairs in which one or both members reported persistent fatigue through patient support group newsletters (60%), clinicians/researchers familiar with CFS (12%), notices placed on electronic bulletin boards for CFS (11%), twin organizations and researchers (6%), relatives and friends (3%) and other sources (8%). Complete data are available for 177 pairs (87%). Twins completed an extensive questionnaire booklet that included measures of physical and mental health, functional status, and psychosocial factors; a structured psychiatric interview was also conducted by telephone. Twins were classified using three increasingly more stringent diagnostic criteria for chronic fatigue: 1) > or = 6 months of fatigue (115 discordant and 61 concordant pairs); 2) chronic fatigue with additional symptoms and application of the medial exclusions of the CDC CFS case definition as obtained by self-report (92 discordant and 41 concordant pairs) and; 3) chronic fatigue with additional symptoms unexplained by self-reported medical conditions and psychiatric diagnoses as determined by the structured interview (69 discordant pairs and 25 concordant pairs). Despite the limitations of a volunteer registry, the Chronic Fatigue Twin Registry promises to be an important resource for research on CFS and chronic fatigue.

Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA.

The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduced and activated intracellularly, producing cytotoxic semiquinone anion radical and hydroquinone reduction intermediates. In vitro, MCRA protects DNA from cross-linking by the hydroquinone reduction intermediate of these mitomycins by oxidizing the hydroquinone back to the parent molecule; thus, MCRA acts as a hydroquinone oxidase. These findings suggest potential therapeutic applications for MCRA in the treatment of cancer with the mitomycins and imply that intrinsic or selected mitomycin C resistance in mammalian cells may not be due solely to decreased bioactivation, as has been hypothesized previously, but instead could involve an MCRA-like mechanism.

Hypoxia-selective nitrobenzyloxycarbonyl derivatives of 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazines.

Some 4- and 2-(nitrobenzyloxycarbonyl)-1, 2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazines (4, 6, and 7) were synthesized and evaluated for their ability to exert preferential toxicity to hypoxic EMT6 mammary carcinoma cells using a colony-forming assay. Of these, the 4,5-dimethoxy-2-nitro analogue 6 (50 microM, 1-h exposure) caused greater than 3 logs of kill of hypoxic cells, with relatively minor toxicity to corresponding aerobic cells. The ability of 4-nitro (4) and 4,5-dimethoxy-2-nitro (6) analogues to reach and kill hypoxic cells of solid tumors was also demonstrated using intradermally implanted EMT6 solid tumors in mice. In addition, a possible source of toxicity to normal tissue, i. e., the activation of the 4-nitrobenzyl derivative 4 by glutathione S-transferase-catalyzed thiolysis, was essentially eliminated by replacing one of the benzylic methylene protons by a methyl group. The 4-nitro (4) and 4,5-dimethoxy-2-nitro (6) analogues also appear to be reduced more easily under acidic conditions (pH 6.0) than under neutral conditions, as measured by differential pulse polarography. Since the pH in hypoxic regions is often lower than that in adjacent aerobic regions, this property should aid in the cytotoxic action of these agents against hypoxic cells of solid tumors.

Exploring the mechanistic aspects of mitomycin antibiotic bioactivation in Chinese hamster ovary cells overexpressing NADPH:cytochrome C (P-450) reductase and DT-diaphorase.

We have directly demonstrated the involvement of human NADPH: cytochrome c (P-450) reductase in the aerobic/hypoxic differential toxicity of mitomycin C and porfiromycin in living cells by varying only this enzyme in a transfected cell line. In the same manner, we have implicated rat DT-diaphorase in the aerobic and hypoxic activation of mitomycin C, but found only a minor role for this enzyme in the aerobic activation of porfiromycin. DT-Diaphorase does not cause the production of an aerobic/hypoxic differential toxicity by mitomycin C, but rather activates this agent through an oxygen insensitive pathway. The evidence suggests that DT-diaphorase activates mitomycin C more effectively than porfiromycin, with porfiromycin being preferentially activated through a one-electron reductive pathway. The therapeutic potential of mitomycin antibiotics in the treatment of cancer can be envisioned to be enhanced for those tumors containing elevated levels of the bioreductive enzymes. However, cytogenetic heterogeneity within the tumor cell population and the various environmental factors which impact on bioreductive enzyme function, including pH and oxygen tension, may subvert this approach. Moreover, if high tumor levels of a drug activating enzyme reflect high levels in the normal tissues of the patient, normal tissue damage may also be enhanced with possibly no improvement in the therapeutic ratio. Approaches utilizing gene therapy, whereby a specific bioreductive catalyst is introduced into the tumor cell population via a targeting vehicle to activate a particular prodrug, may be more effective in that not only will the prodrug of choice be specifically activated in the tumor, but the source of the catalyst, be it bacterial, rodent, or human, will not be important. In fact, in the case of DT-diaphorase and mitomycin C, the rat form of the enzyme could be advantageous because it is more effective in activating mitomycin C than is the human form of this enzyme. Assuming targeted gene delivery to malignant cells, a non-host enzyme which is more effective at activating mitomycin C than the analogous host enzyme might also result in less drug activation in normal tissue and, hence, less normal tissue toxicity.

The intracellular location of NADH:cytochrome b5 reductase modulates the cytotoxicity of the mitomycins to Chinese hamster ovary cells.

NADH:cytochrome b5 reductase activates the mitomycins to alkylating intermediates in vitro. To investigate the intracellular role of this enzyme in mitomycin bioactivation, Chinese hamster ovary cell transfectants overexpressing rat NADH:cytochrome b5 reductase were generated. An NADH:cytochrome b5 reductase-transfected clone expressed 9-fold more enzyme than did parental cells; the levels of other mitomycin-activating oxidoreductases were unchanged. Although this enzyme activates the mitomycins in vitro, its overexpression in living cells caused decreases in sensitivity to mitomycin C in air and decreases in sensitivity to porfiromycin under both air and hypoxia. Mitomycin C cytotoxicity under hypoxia was similar to parental cells. Because NADH:cytochrome b5 reductase resides predominantly in the mitochondria of these cells, this enzyme may sequester these drugs in this compartment, thereby decreasing nuclear DNA alkylations and reducing cytotoxicity. A cytosolic form of NADH:cytochrome b5 reductase was generated. Transfectants expressing the cytosolic enzyme were restored to parental line sensitivity to both mitomycin C and porfiromycin in air with marked increases in drug sensitivity under hypoxia. The results implicate NADH:cytochrome b5 reductase in the differential bioactivation of the mitomycins and indicate that the subcellular site of drug activation can have complex effects on drug cytotoxicity.

Formation of a major DNA adduct of the mitomycin metabolite 2,7-diaminomitosene in EMT6 mouse mammary tumor cells treated with mitomycin C.

Treatment of EMT6 mouse mammary tumor cells with [3H]mitomycin C (MC) results in the formation of six major DNA adducts, as described earlier using an HPLC assay of 3H-labeled products of enzymatic hydrolysis of DNA isolated from MC-treated cells. Four of these adducts were identified as monofunctional and bifunctional guanine-N2 adducts in the minor groove of DNA. In order to establish relationships between individual types of MC-DNA adducts and biological responses it is necessary to identify all of the adducts formed in cells. To this end we have now identified a predominant, previously unknown adduct formed in MC-treated EMT6 cells as a derivative not of MC, but of 2,7-diaminomitosene (2,7-DAM), the major bioreductive metabolite of MC. Rigorous proof demonstrates that it is a DNA major groove, guanine-N7 adduct of 2,7-DAM, linked at C-10 to DNA. The adduct is relatively stable at ambient temperature, but is readily depurinated upon heating. Its isolation from MC-treated cells indicates that MC is reductively metabolized to 2,7-DAM, which then undergoes further reductive activation to alkylate DNA, along with the parent MC. Low MC:DNA ratios were identified as a critical factor promoting 2,7-DAM adduct formation in an in vitro model calf thymus DNA/ MC/reductase model system, as well as in MC-treated EMT6 cells. The 2,7-DAM-guanine-N7 DNA adduct appears to be relatively noncytotoxic, as indicated by the dramatically lower cytotoxicity of 2,7-DAM in comparison with MC in EMT6 cells. Like MC, 2,7-DAM exhibited slightly greater cytotoxicity to cells treated under hypoxic as compared to aerobic conditions. However, 2,7-DAM was markedly less cytotoxic than MC under both aerobic and hypoxic conditions. Thus, metabolic reduction of MC to 2,7-DAM represents a detoxification process. The differential effects of MC-DNA and 2,7-DAM-DNA adducts support the concept that specific structural features of the DNA damage may play a critical role in the cytotoxic response to a DNA-targeted chemotherapeutic agent.

Bioactivation of mitomycin antibiotics by aerobic and hypoxic Chinese hamster ovary cells overexpressing DT-diaphorase.

DT-Diaphorase catalyzes a two-electron reduction of mitomycin C (MC) and porfiromycin (POR) to reactive species. Many cell lines that overexpress DT-diaphorase and are sensitive to the mitomycins are protected from the aerobic cytotoxicity of these drugs by the DT-diaphorase inhibitor dicumarol. The cytoprotective properties of this relatively non-specific inhibitor, however, vanish under hypoxic conditions. To ascertain the role of DT-diaphorase in mitomycin bioactivation and cytotoxicity in living cells, a rat liver DT-diaphorase cDNA was transfected into Chinese hamster ovary cells. MC was equitoxic to the parental cells under oxygenated and hypoxic conditions. In contrast, POR was less toxic than MC to these cells under aerobic conditions, but significantly more toxic than MC under hypoxia. Two DT-diaphorase-transfected clones displayed increases in DT-diaphorase activity of 126- and 133-fold over parental cells. The activities of other oxidoreductases implicated in mitomycin bioreduction were unchanged. MC was more toxic to both DT-diaphorase-transfected lines than to parental cells; the toxicity of MC to the transfected lines was similar in air and hypoxia. POR was also more toxic to the DT-diaphorase-elevated clones than to parental cells under oxygenated conditions. Under hypoxia, however, the toxicity of POR to the transfected clones was unchanged from that of parental cells. The findings implicate DT-diaphorase in mitomycin bioactivation in living cells, but suggest that this enzyme does not contribute to the differential toxicity of MC or POR in air and hypoxia.

Differential toxicity of mitomycin C and porfiromycin to aerobic and hypoxic Chinese hamster ovary cells overexpressing human NADPH:cytochrome c (P-450) reductase.

Purified NADPH:cytochrome c (P-450) reductase (FpT; NADPH-ferrihemoprotein oxidoreductase, EC 1.6.2.4) can reductively activate mitomycin antibiotics through a one-electron reduction to species that alkylate DNA. To assess the involvement of FpT in the intracellular activation of the mitomycins, transfectants overexpressing a human FpT cDNA were established from a Chinese hamster ovary cell line deficient in dihydrofolate reductase (CHO-K1/dhfr-). The parental cell line was equisensitive to the cytotoxic action of mitomycin C under oxygenated and hypoxic conditions. In contrast, porfiromycin was considerably less cytotoxic to wild-type parental cells than was mitomycin C in air and markedly more cytotoxic under hypoxia. Two FpT-transfected clones were selected that expressed 19- and 27-fold more FpT activity than the parental line. Levels of other oxidoreductases implicated in the activation of the mitomycins were unchanged. Significant increases in sensitivity to mitomycin C and porfiromycin in the two FpT-transfected clones were seen under both oxygenated and hypoxic conditions, with the increases in toxicity being greater under hypoxia than in air. These findings demonstrate that FpT can bioreductively activate the mitomycins in living cells and implicate FpT in the differential aerobic/hypoxic toxicity of the mitomycins.

Preferential kill of hypoxic EMT6 mammary tumor cells by the bioreductive alkylating agent porfiromycin.

Hypoxic cells in solid tumors represent a therapeutically resistant population that limits the curability of many solid tumors by irradiation and by most chemotherapeutic agents. The oxygen deficit, however, creates an environment conducive to reductive processes; this results in a major exploitable difference between normal and neoplastic tissues. The mitomycin antibiotics can be reductively activated by a number of oxidoreductases, in a process required for the production of their therapeutic effects. Preferential activation of these drugs under hypoxia and greater toxicity to oxygen-deficient cells than to their oxygenated counterparts are obtained in most instances. The demonstration that mitomycin C and porfiromycin, used to kill the hypoxic fraction, in combination with irradiation, to eradicate the oxygenated portion of the tumor, produced enhanced cytodestructive effects on solid tumors in animals has led to the clinical evaluation of the mitomycins in combination with radiation therapy in patients with head and neck cancer. The findings from these clinical trials have demonstrated the value of directing a concerted therapeutic attack on the hypoxic fraction of solid tumors as an approach toward enhancing the curability of localized neoplasms by irradiation.

Mitomycin C: a prototype bioreductive agent.

Hypoxic cells of solid tumors represent a therapeutically resistant population that limits the curability of many solid tumors by x-irradiation and by most chemotherapeutic agents. The oxygen deficit, however, creates an environment conducive to reductive processes that results in a major exploitable difference between normal and neoplastic tissues. Mitomycin C (MC) can be reductively activated by a number of oxidoreductases, in a process required for the production of its therapeutic effects. This enzymatic reduction results in preferential activation of MC under hypoxia and, in most instances, the production of greater toxicity to oxygen-deficient cells than to their oxygenated counterparts. DNA appears to be the most important target of the reactive species generated from MC, with both mono- and bis-adducts of DNA being formed in drug-treated cells. The demonstration that MC, used to kill the hypoxic fraction, in combination with x-irradiation, to eradicate the oxygenated portion of the tumor, produced enhanced cytodestructive effects on solid tumors of animals has led to the clinical evaluation of the mitomycin antibiotics in combination with x-rays in patients with cancers of the head and neck. The findings from these clinical trials have demonstrated the utility of directing a concerted therapeutic attack on the hypoxic fraction of solid tumors as an approach toward enhancing the curability of localized neoplasms by x-irradiation.

Ribosomal frameshifting in the yeast retrotransposon Ty: tRNAs induce slippage on a 7 nucleotide minimal site.

Ribosomal frameshifting regulates expression of the TYB gene of yeast Ty retrotransposons. We previously demonstrated that a 14 nucleotide sequence conserved between two families of Ty elements was necessary and sufficient to support ribosomal frameshifting. This work demonstrates that only 7 of these 14 nucleotides are needed for normal levels of frameshifting. Any change to the sequence CUU-AGG-C drastically reduces frameshifting; this suggests that two specific tRNAs, tRNA(UAGLeu) and tRNA(CCUArg), are involved in the event. Our tRNA overproduction data suggest that a leucyl-tRNA, probably tRNA(UAGLeu), an unusual leucine isoacceptor that recognizes all six leucine codons, slips from CUU-Leu onto UUA-Leu (in the +1 reading frame) during a translational pause at the AGG-Arg codon induced by the low availability of tRNA(CCUArg), encoded by a single-copy essential gene. Frameshifting is also directional and reading frame specific. Interestingly, frameshifting is inhibited when the "slip" CUU codon is located three codons downstream, but not four or more codons downstream, of the translational initiation codon.

Enhancer and silencerlike sites within the transcribed portion of a Ty2 transposable element of Saccharomyces cerevisiae.

The Ty2-917 element is a member of the Ty2 class of retroviruslike transposable elements of Saccharomyces cerevisiae. We showed that regions downstream of the Ty2-917 transcription start site modulate its transcription. One region was located downstream of the transcription initiation site (position 240) and within the first 559 base pairs of the element. This region had a dramatic effect, causing an approximately 1,000-fold increase in steady-state levels of RNA. The region stimulated transcription when placed in either orientation upstream of a heterologous gene, HIS4, lacking its own upstream activation sequence (UAS). We termed this positively acting region an enhancer, by analogy to sites described in higher cells, to distinguish it from yeast UASs which do not function when placed within the transcribed portion of the gene. Though, like some higher eucaryotic enhancers, the Ty2-917 enhancer is located within the transcribed region, it is unlike them in that it occurs within a coding region rather than in an intron. The Ty2-917 enhancer and the Ty2-917 UAS had a synergistic effect on transcription, together stimulating transcription 15-fold over the predicted additive effect. We also identified a site which decreases RNA accumulation, located about 750 base pairs into the element. This site functioned in only one orientation when inserted upstream of the UAS-less heterologous gene. The site was similar to silencers, or negative enhancers, in that it acted to repress transcription from outside the transcribed region, but was distinct in that the function of a canonical silencer was independent of orientation.