The where, what and how of ribosomal frameshifting in retroviral protein synthesis.

The gag and pol genes of most retroviruses occur in different reading frames and their translation as a single polypeptide is carried out by ribosomal frameshifting in the -1 direction. The alignment of the different reading frames occurs by overlapping reading in response to at least two signals within the RNA: one is a heptanucleotide stretch at the frameshift site and the other is a stem-loop structure which occurs just downstream of the first signal.

Hormonal responses to a 160-km race across frozen Alaska.

BACKGROUND: Severe physical and environmental stress seems to have a suppressive effect on the hypothalamic-pituitary-gonadal (HPG) axis in men. Examining hormonal responses to an extreme 160-km competition across frozen Alaska provides a unique opportunity to study this intense stress. OBJECTIVE: To examine hormonal responses to an ultra-endurance race. METHODS: Blood samples were obtained from 16 men before and after racing and analyzed for testosterone, interleukin-6 (IL-6), growth hormone (GH) and cortisol. Six subjects (mean (SD) age 42 (7) years; body mass 78.9 (7.1) kg; height 1.78 (0.05) m raced by bicycle (cyclists) and 10 subjects (age 35 (9) years; body mass 77.9 (10.6) kg; height, 1.82 (0.05) m) raced by foot (runners). Mean (SD) finish times were 21.83 (6.27) and 33.98 (6.12) h, respectively. RESULTS: In cyclists there were significant (p< or =0.05) mean (SD) pre-race to post-race increases in cortisol (254.83 (135.26) to 535.99 (232.22) nmol/l), GH (0.12 (0.23) to 3.21 (3.33) microg/ml) and IL-6 (2.36 (0.42) to 10.15 (3.28) pg/ml), and a significant decrease in testosterone (13.81 (3.19) to 5.59 (3.74) nmol/l). Similarly, in runners there were significant pre-race to post-race increases in cortisol (142.09 (50.74) to 452.21 (163.40) ng/ml), GH (0.12 (0.23) to 3.21 (3.33) microg/ml) and IL-6 (2.42 (0.68) to 12.25 (1.78) pg/ml), and a significant decrease in testosterone (12.32 (4.47) to 6.96 (3.19) nmol/l). There were no significant differences in the hormonal levels between cyclists and runners (p>0.05). CONCLUSIONS: These data suggest a suppression of the hypopituitary-gonadal axis potentially mediated by amplification of adrenal stress responses to such an ultra-endurance race in environmentally stressful conditions.

Selenocysteine tRNA[Ser]Sec gene is ubiquitous within the animal kingdom.

Recently, a mammalian tRNA which was previously designated as an opal suppressor seryl-tRNA and phosphoseryl-tRNA was shown to be a selenocysteyl-tRNA (B. J. Lee, P. J. Worland, J. N. Davis, T. C. Stadtman, and D. Hatfield, J. Biol. Chem. 264:9724-9727, 1989). Hence, this tRNA is now designated as selenocysteyl-tRNA[Ser]Sec, and its function is twofold, to serve as (i) a carrier molecule upon which selenocysteine is biosynthesized and (ii) as a donor of selenocysteine, which is the 21st naturally occurring amino acid of protein, to the nascent polypeptide chain in response to specific UGA codons. In the present study, the selenocysteine tRNA gene was sequenced from Xenopus laevis, Drosophila melanogaster, and Caenorhabditis elegans. The tRNA product of this gene was also identified within the seryl-tRNA population of a number of higher and lower animals, and the human tRNA[Ser]Sec gene was used as a probe to identify homologous sequences within genomic DNAs of organisms throughout the animal kingdom. The studies showed that the tRNA[Ser]Sec gene has undergone evolutionary change and that it is ubiquitous in the animal kingdom. Further, we conclude that selenocysteine-containing proteins, as well as the use of UGA as a codon for selenocysteine, are far more widespread in nature than previously thought.

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.

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.

Aminoacyl-transfer RNA populations in mammalian cells chromatographic profiles and patterns of codon recognition.

The chromatographic profiles of 20 aminoacyl-tRNAs from rabbit liver were compared to those of rabbit reticulocytes by reverse phase chromatography and the chromatographic profiles of 20-aminoacyl-tRNAs from bovine liver were compared to those of bovine brain. The two rabbit tissues showed significant differences in the elution profiles of most aminoacyl-tRNAs, while the elution profiles of the aminoacyl-tRNAs from the bovine tissues showed fewer differences. The patterns of codon recognition of several aminoacyl-tRNAs fractionated from rabbit reticulocytes have also been compared to those fractionated from rabbit liver.

Replenishment of selenium deficient rats with selenium results in redistribution of the selenocysteine tRNA population in a tissue specific manner.

We reported previously that the selenium status of rats influences both the steady-state levels and distributions of two selenocysteine tRNA isoacceptors and that these isoacceptors differ by a single methyl group attached to the ribosyl moiety at position 34. In this study, we demonstrate that repletion of selenium-deficient rats results in a gradual, tissue-dependent shift in the distribution of these isoacceptors. Rats fed a selenium-deficient diet possess a greater abundance of the species unmethylated on the ribosyl moiety at position 34 compared to the form methylated at this position. A redistribution of the Sec-tRNA isoacceptors occurred in tissues of selenium-supplemented rats whereby the unmethylated form gradually shifted toward the methylated form. This was true in each of four tissues examined, muscle, kidney, liver and heart, although the rate of redistribution was tissue-specific. Muscle manifested a predominance of two minor serine isoacceptors under conditions of extreme selenium-deficiency which also appeared to respond to selenium. Ribosomal binding studies revealed that one of the two additional isoacceptors decodes the serine codeword, AGU, and the second decodes the serine codeword, UCU. Interestingly, muscle and heart were the slower tissues to return to a 'selenium adequate' tRNA distribution pattern.

Effects of post-transcriptional base modifications on the site-specific function of transfer RNA in eukaryote translation.

The site-specific function in translation of several naturally occurring mammalian transfer RNAs has been studied in a series of investigations with some similarities to studies in other laboratories of tRNAs in suppression. Equal amounts of aminoacyl-tRNA isoacceptors with contrasting isotopes were added in pairs to reticulocyte lysates and allowed to incorporate their amino acids into rabbit globin. Rates of incorporation from unlimiting amounts of each isoacceptor into the corresponding amino-acid-containing sites were determined. The tRNAs of each isoacceptor pair differed as to post-transcriptional base modifications. The natural occurrence of these isoacceptors can be correlated with rates of cellular division, with more rapidly dividing and neoplastic cells containing hypomodified tRNA. The overall incorporation of lysine into globin from a fully modified tRNALys that decodes AAG is faster by 25 to 30% than from the corresponding hypomodified tRNALys. There is considerable scatter in values for incorporation ratios at different lysine-containing sites, with the hypomodified isoacceptor even being preferred at one site. The AAG decoding isoacceptors are capable of translating AAA although much more slowly than AAG. In translating AAA, in contrast to translating AAG, the hypomodified tRNALys isoacceptor is preferred. A Y base-deficient hypomodified tRNAPhe isoacceptor found only in some kinds of rapidly dividing tumor cells donates its phenylalanine preferentially to globin in competition with the fully modified Y-containing tRNAPhe of liver by 15 to 17%. There is a considerable range of incorporation ratios at the different phenylalanine-containing sites of the globin subunits. No correlation can be made between the isoacceptor preferred and the phenylalanine codon being translated. The incorporation of histidine from a fully modified tRNAHis-containing Q base in its anticodon, compared with that from the hypomodified counterpart isoacceptor that lacks Q base and that occurs in rapidly dividing cells, showed no difference in their ability to incorporate overall or into individual histidine-containing sites. There is little evidence that adjacent bases or codons in messenger RNA affect the tRNAs preferred in the translation of most sites. A striking pattern of tRNA preference was observed in three cases in which there are tandem codons, with the same codon appearing twice in succession.(ABSTRACT TRUNCATED AT 400 WORDS)

Ischaemia induced development of functional coronary collateral circulation in ponies.

The response of coronary collaterals in nine ponies subjected to repeated reversible occlusions (2 min duration, 30 min interval) of the left anterior descending coronary artery was studied at rest. Each pony was instrumented with a Doppler flowmeter and hydraulic cuff occluder around the left anterior descending coronary artery, left ventricular subendocardial sonomicrometers, and a left ventricular micromanometer. Initial occlusions increased end diastolic myocardial segment length by 3% and decreased segment systolic shortening, stroke work, and velocity of shortening by 103%, 95%, and 79% respectively in the left ventricular apex. Left ventricular systolic and end diastolic pressure, peak positive dP/dt, and heart rate were not significantly changed by occlusion. After 421(70) (mean(SEM)) occlusions no sustained alterations in myocardial segment function occurred in response to occlusion. Thus the presence of a subendocardial plexus did not protect against a severe loss of myocardial segment function when the ponies were initially subjected to occlusions of the left anterior descending coronary artery. However, repeated reversible occlusions enhanced coronary collateral blood flow such that it was adequate to maintain left ventricular function in the absence of left anterior descending coronary artery flow. It is concluded that the pony is highly suitable for use in studies of coronary collateral circulation because of its coronary anatomical similarity to man and its capacity to develop functional collateralisation.

A pseudogene for human glutathione peroxidase.

Glutathione peroxidases (GPx) serve a bioprotective function in the reduction of peroxides to less toxic substances. Both cellular and secreted forms of the protein have been reported, as well a number of distinct cDNA sequences. Previous efforts have described three distinct loci on human chromosomes 3, 21 and X which hybridize to a GPX cDNA and these authors have speculated that only the chromosome 3 locus encodes a functional GPX gene. This conclusion was based on mapping studies showing a precise deletion of intron sequences in the GPX loci on chromosomes 21 and X despite strong conservation among these sequences in both the coding and 3'-untranslated regions. To pursue this issue, we have isolated the chromosome 21 GPX locus by molecular cloning and determined its nucleotide sequence. Consistent with the expectations of McBride et al. [Biofactors 4 (1988) 285-292], the sequence does reveal a highly conserved processed pseudogene. It is suggested that a retrotransposed copy of the GPX gene integrated into chromosome 21 and may have maintained activity prior to the accumulation of inactivating mutations.

Differential mode of TBP utilization in transcription of the tRNA[Ser]Sec gene and TATA-less class III genes.

The Xenopus laevis selenocysteine tRNA[Ser]Sec gene utilizes the TATA box binding protein (TBP) for its transcription in a manner more like TATA-dependent class II genes than TATA-less class III tRNA genes, even though this gene is transcribed by RNA polymerase III (Pol III). Addition of TBP increased in vitro transcription of the tRNA[Ser]Sec gene and a RNA polymerase II-(Pol II-) dependent template, while it decreased TATA-independent tRNA(Met) gene transcription, in a dose-dependent manner. Addition of wild-type TBP, truncated TBP containing the highly conserved COOH-terminal domain or a mutant TBP defective in TATA-independent Pol III transcription to TBP-depleted extracts restored tRNA[Ser]Sec transcription, while addition of a mutant TBP defective in Pol II transcription did not. These studies provide evidence that common surfaces of TBP may be used in transcription from TATA-dependent promoters of the tRNA[Ser]Sec gene and class II genes. Further, we show that distinct chromatographic fractions of TBP complexes are required for tRNA[Ser]Sec gene transcription and TATA-less class III gene transcription.

Upstream promoter elements are sufficient for selenocysteine tRNA[Ser]Sec gene transcription and to determine the transcription start point.

The TATA box, located upstream at about nt -30, and the proximal sequence element, located at about nt -60, are both essential and sufficient for basal level transcription of the Xenopus laevis (Xl) selenocysteine (Sec) tRNA[Ser]Sec gene as demonstrated by its microinjection into Xl oocytes. Point mutations within either of these regions abolish transcription, while deletion of the internal boxA element or insertion of 13 nt within the internal boxB element does not impair transcription. The latter mutations (within the internal regions) affect processing of the 3'-trailer sequence. Replacement of the tRNA[Ser]Sec coding sequence with an Escherichia coli M1 RNA gene resulted in expression of the E. coli gene governed by the upstream tRNA[Ser]Sec promoter elements. These studies demonstrate unequivocally that the upstream promoter elements are sufficient for the basal level of tRNA[Ser]Sec gene transcription. Primer extension studies with spacer mutants show that the internal elements do not play a role in selecting the transcription start point (tsp), but that selection of the tsp is determined by the region upstream from the gene. Further, studies with spacer mutants show that the distance between the TATA box and the tsp is quite likely the critical factor in selecting the position of tsp.

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.

The zebrafish genome contains two distinct selenocysteine tRNA[Ser]sec genes.

The zebrafish is widely used as a model system for studying mammalian developmental genetics and more recently, as a model system for carcinogenesis. Since there is mounting evidence that selenium can prevent cancer in mammals, including humans, we characterized the selenocysteine tRNA[Ser]sec gene and its product in zebrafish. Two genes for this tRNA were isolated and sequenced and were found to map at different loci within the zebrafish genome. The encoding sequences of both are identical and their flanking sequences are highly homologous for several hundred bases in both directions. The two genes likely arose from gene duplication which is a common phenomenon among many genes in this species. In addition, zebrafish tRNA[Ser]sec was isolated from the total tRNA population and shown to decode UGA in a ribosomal binding assay.

Comparison of the codon recognition properties and of the utilization of normal and tumor specific Phe-tRNAs in protein synthesis.

Phe-tRNA from normal rat liver (designated Phe-tRNAN) and the under-modified, tumor specific, Phe-tRNAs from mouse neuroblastoma (designated Phe-tRNANB) and rat lymphoma (designated Phe-tRNARL) recognize the phenylalanine codons, UUU and UUC in a ribosome binding assay, but not other codons that differ from UUU and UUC in a single base at either the 5' or 3' position. Phe-tRNANB was incorporated into protein more extensively than either Phe-tRNARL or Phe-tRNAN in wheat germ extracts programmed with globin mRNA. The utilization level of each Phe-tRNA was correlated with its rate of deacylation in wheat germ extracts, i.e., Phe-tRNANB deacylated less rapidly than Phe-tRNARL or Phe-tRNAN. Phe-tRNA, from which the Y base was chemically excised (designated Phe-tRNA-Y), did not respond to UUU or UUC in the ribosomal binding assay, nor did it transfer its phenylalanine to protein in wheat germ extracts programmed with globin mRNA.

Selenium supplementation suppresses tumor necrosis factor alpha-induced human immunodeficiency virus type 1 replication in vitro.

Selenium is a nutritionally essential trace element that is important for optimal function of the immune system. It is incorporated into selenoproteins as the amino acid selenocysteine and it is known to inhibit the expression of some viruses. In this study, we show that selenium supplementation for 3 days prior to exposure to tumor necrosis factor alpha (TNF-alpha) partially suppresses the induction of human immunodeficiency virus type 1 (HIV-1) replication in both chronically infected T lymphocytic and monocytic cell lines. In acute HIV-1 infection of T lymphocytes and monocytes in the absence of exogenous TNF-alpha, the suppressive effect of selenium supplementation was not observed. However, selenium supplementation did suppress the enhancing effect of TNF-alpha on HIV-1 replication in vitro in acutely infected human monocytes, but not in T lymphocytes. Selenium supplementation also increased the activities of the selenoproteins, glutathione peroxidase (GPx) and thioredoxin reductase (TR), which serve as cellular antioxidants. Taken together, these results suggest that selenium supplementation may prove beneficial as an adjuvant therapy for AIDS through reinforcement of endogenous antioxidative systems.

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.

Hypoplastic aortoiliac syndrome: An entity peculiar to women.

Hypoplastic aortoiliac syndrome (HAIS) is a unique disorder that occurs almost exclusively in women. Because reports in the literature are scant, we reviewed our own experience. The criteria for definition were high bifurcation of the abdominal aorta, straight course of the iliac arteries without the normal characteristic bowing, acute angle of the aortic bifurcation, aortic diameter of 14 mm or less, and iliac artery diameter of 7 mm or less. Nineteen patients with HAIS were identified in a 14-year period. All 19 patients were heavy smokers. Most had significant associated arterial disease, and all were women. Fifteen patients were selected as candidates for operation. Two were treated conservatively, one refused surgery, and one died of a myocardial infarction before operation. Reconstruction was performed by aortoiliac thromboendarterectomy with vein patch for segmental disease (four patients) and Dacron bifurcating graft for diffuse disease (11 patients). There were no deaths during operation. Four patients required reoperations for graft failure and three patients eventually required amputations. The four patients who underwent reconstructions by thromboendarterectomy with vein patch had uniformly good results. From this experience, the following approach and management are recommended: conservative nonoperative exercise program with close follow-up if symptoms are tolerable, aortoiliac thromboendartectomy and vein patch if disease is segmental, aortofemoral bifurcating graft for patients with diffuse disease with graft size closely approximating vessel size, and concomitant femoral-to-popliteal graft to ensure adequate runoff and graft patency if there is significant femoral disease. The etiology of HAIS remains obscure. Early onset of symptoms seems to be due to small vessels with superimposed atherosclerotic plaques. Addiction to smoking in these women is extreme (paralleling that seen in Buerger's disease) and may be of etiologic significance.

Cardiac cycle length variability in ponies at rest and during exercise.

We evaluated cardiac cycle length variability in ponies at rest and during strenuous exercise with and without premedication with atropine. In the absence of premedication, cardiac cycle length at rest was 1,112 +/- 53 ms, the individual cardiac cycle length standard deviation (SDCL) was 75 +/- 23 ms, and the individual cycle length coefficient of variation (CVCL) was 6.32 +/- 1.62. Exercise significantly decreased (P < 0.05) all three indexes (290 +/- 9 ms, 5 +/- 1 ms, and 1.65 +/- 0.20, respectively). Atropine premedication significantly reduced resting cardiac cycle length (685 +/- 46 ms), SDCL (10 +/- 2 ms), and CVCL (1.45 +/- 0.19) compared with nonpremedicated values. Cardiac cycle length was significantly decreased by exercise after atropine premedication, but no statistically significant changes occurred in SDCL or CVCL. Thus, although considerable cardiac cycle length variability exists in nonpremedicated ponies at rest, it is nearly completely abolished by strenuous exercise. The absence of significant differences between the indexes of variability during exercise without premedication, at rest after atropine, and during exercise after atropine indicates that cardiac cycle length variability in the pony is mediated primarily through activity of the parasympathetic system.

Yeast asparagine (Asn) tRNA without Q base promotes eukaryotic frameshifting more efficiently than mammalian Asn tRNAs with or without Q base.

In this study, we compare the efficiency of Asn tRNA from mammalian sources with and without the highly modified queuosine (Q) base in the wobble position of its anticodon and Asn tRNA from yeast, which naturally lacks Q base, to promote frameshifting. Interestingly, no differences in the ability of the two mammalian Asn tRNAs to promote frameshifting were observed, while yeast tRNA(ASn)(-Q) promoted frameshifting more efficiently than its mammalian counterparts in both rabbit reticulocyte lysates and wheat germ extracts. The shiftability of yeast Asn tRNA is therefore not due, or at least not completely, to the lack of Q base and most likely the shiftiness resides in structural differences elsewhere in the molecule. However, we cannot absolutely rule out a role of Q base in frameshifting as wheat germ extracts and a lysate depleted of most of its tRNA and supplemented with calf liver tRNA contain both Asn tRNA with or without Q base.