Serine insertion caused by the ribosomal suppressor SUP46 in yeast

The ribosomal suppressor SUP46 isolated from the yeast Saccharomyces cerevisiae suppresses a broad range of mutations, including at least some UAA, UAG and UGA alleles. The SUP46 suppressor causes the insertion of serine into iso-1-cytochrome c at the site of the UAA mutation in the cyc1-72 allele. It is believed that the altered ribosomes in the SUP46 suppressor allow a serine tRNA to misread UAA codons.     

Substitution of serine caused by a recessive lethal suppressor in yeast.

The SUP-RL1 suppressor in the yeast Saccharomyces cerevisiae causes lethality in haploid strains but not in diploid or aneuploid strains that are heterozygous for the suppressor locus. This recessive lethal suppressor acts on amber (UAG) nutritional markers, and can cause the production of approximately 50% of the normal amount of iso-1-cytochrome c in disomic strains that are heterozygous for the SUP-RL1 suppressor, and that contain the cyc1-179 allele which has an amber codon corresponding to amino acid position 9. The suppressed iso-1-cytochrome c contains a residue of serine at the position that corresponds to the site of the amber codon. SUP-RL1 was found to lie between thr4 and MAL2 on chromosome III, approximately 30 map units from the mating-type locus. It is suggested that the gene product of SUP-RL1 may be a species of serine transfer RNA that normally reads the serine codon UCG, and that is represented only once in the haploid genome.     

Synthesis of an ochre suppressor tRNA gene and expression in mammalian cells.

We have used site-specific mutagenesis to change the anticodon of a Xenopus laevis tyrosine tRNA gene so that it would recognize ochre codons. This tRNA gene is expressed when amplified in monkey cells as part of a SV40 recombinant and efficiently suppresses termination at both the ochre codon separating the adenovirus 2 hexon gene from a 23-kd downstream gene and the ochre codon at the end of the NS1 gene of influenza virus A/Tex/1/68. Termination at an amber codon of a NS1 gene of another influenza virus strain was not suppressed by the (Su+) ochre gene suggesting that in mammalian cells amber codons are not recognized by ochre suppressor tRNAs. Finally, microinjection into mammalian cells of both (Su+) ochre tRNA genes and selectible genes containing ochre nonsense mutations gives rise to colonies under selective conditions. We conclude that it should be possible to isolate a wide assortment of mammalian cell lines with ochre suppressor activity.     

Mutagenesis in Escherichia coli. V. Attempted interconversion of ochre and amber suppressors and mutational instability due to an ochre suppressor

Summary A possible quantitative system for the interconversion of ochre and amber suppressors was studied in Escherichia coli WU36-10, a strain in which a leucine requirement is suppressed by amber suppressors and a tyrosine requirement is suppressed by ochre suppressors. The conversion of am Sup-2⁺ to oc Sup-2⁺ occurred at rates similar to those for the de novo induction of such suppressors, both spontaneously and after ultraviolet or gamma irradiation. Both induction and conversion of suppressors showed the phenomenon of mutation frequency decline after ultraviolet light. Conversions in the opposite direction from oc Sup-2⁺ to am Sup-2⁺ were, however, not detected in unmutagenised populations of oc Sup-2⁺ strains derived either by conversion from an am Sup-2⁺ strain or de novo from the parental WU36-10, nor were they detected after treatment with ultraviolet light, gamma radiation or 2-aminopurine. If the conversion of oc Sup-2⁺ to am Sup-2⁺ occurs at all, it is at a rate very considerably lower than that for the conversion of am Sup-2⁺ to oc Sup-2⁺. Some Tyr⁺ oc Sup-2⁺ mutants demonstrated mutation rates c. 100 times greater than those of WU36-10 for mutation to Leu⁺ spontaneously and after ultraviolet or gamma radiation. Possible explanations of this are discussed.     

supN ochre suppressor gene in Escherichia coli codes for tRNALys.

We describe the cloning and nucleotide sequence of a new tRNALys gene, lysV, in Escherichia coli. An ochre suppressor allele of this gene, supN, codes for a tRNALys with anticodon UUA, presumably derived by a single base change from a wild-type UUU anticodon. The sequence of the supN tRNALys is identical to the sequence of ochre suppressor tRNAs encoded by mutant alleles at the lysT locus. This locus, which contains the two previously known tRNALys genes of E. coli, is located far from the lysV locus on the chromosome.     

The Bacillus subtilis ochre suppressor sup-3 is located in an operon of seven tRNA genes.

Most Bacillus subtilis tRNA genes have been isolated from lambda libraries by use of probes that hybridize to tRNA or rRNA sequences. None of those genes map to the region of the sup-3 mutation. By cloning of the sup-3 allele, a cluster of seven tRNA genes (the trnS operon) that had not been isolated by other methods was identified. In principle, this approach could be used to isolate at least one more predicted tRNA-containing operon in this bacterium. The trnS operon was shown to contain tRNA genes for Asn (GUU), Ser (GCU), Glu (UUC), Gln (UUG), Lys (UUU), Leu (UAG), and Leu (GAG). The sup-3 mutation was found to be a T-to-A transversion that changes the anticodon of the lysine tRNA from 5'-UUU-3' to 5'-UUA-3'. This result agrees with previous work that determined that the sup-3 mutation causes lysine to be inserted at ochre nonsense mutations.     

Partial suppression of an ochre mutation in Saccharomyces cerevisiae by multicopy plasmids containing a normal yeast tRNAGln gene

We screened a yeast genomic library for recombinant DNA plasmids that complemented the ultraviolet (u.v.) sensitivity of a strain of Saccharomyces cerevisiae designated rad4-3 that is defective in excision repair of DNA. A multicopy plasmid (pNF4000) with a 9.4 X 10(3) base-pair yeast DNA insert partially complemented the u.v. sensitivity of rad4-3, but not of two other rad4 allelic mutants (rad4-2 and rad4-4), or of other u.v.-sensitive rad mutants. The yeast insert was analyzed by restriction mapping, DNA-DNA hybridization, DNA-tRNA hybridization and DNA sequencing. This analysis revealed the presence of a normal tRNAGln gene, a yeast sigma element situated 5' to the transfer RNA gene, a Ty element and a solo delta element. Deletion analysis of pNF4000 showed that the tRNAGln gene is required for partial complementation of the u.v. sensitivity of rad4-3. Furthermore, a multicopy plasmid containing a tRNAGln gene derived from a different region of the yeast genome also partially complemented the u.v. sensitivity of rad4-3. The rad4-3 mutation is suppressed following transformation with a plasmid containing the known ochre suppressor SUP11-o, indicating that it is an ochre mutation. We therefore conclude that when expressed in sufficient quantity, normal tRNAGln (which usually decodes the sense codon CAA) can weakly suppress the nonsense ochre codon UAA, and suggest that this represents an example of wobble occurring at the first rather than at the third position of the codon.     

Isolation of glutamate-inserting ochre suppressor mutants of Salmonella typhimurium and Escherichia coli.

Glutamate-inserting ochre suppressors have been identified among late-arising, spontaneous revertants of a hisG428 mutant of Salmonella typhimurium and an argE3 mutant of Escherichia coli. The S. typhimurium suppressors mapped in the tRNA2(Glu) gene gltU at 82 min; those in E. coli were found to be in tRNA2(Glu) genes gltW at 56 min, gltU at 85 min, and gltT at 90 min.     

Escherichia coli supH suppressor: temperature-sensitive missense suppression caused by an anticodon change in tRNASer2.

We describe the cloning and the DNA sequence of the Escherichia coli supH missense suppressor and of the supD60(Am) suppressor genes. supH is a mutant form of serU which codes for tRNASer2. The supH coding sequence differs from the wild-type sequence by a single nucleotide change which corresponds to the middle position of the anticodon. The CGA anticodon of wild-type tRNA and CUA anticodon of supD tRNA is changed to CAA in supH tRNA, which is expected to recognize the UUG leucine codon. We propose that the supH suppressor causes the insertion of serine in response to this codon. The temperature sensitivity caused by supH may be due to a conformation of the CAA anticodon in the supH tRNASer that is slightly different than that in the corresponding tRNALeu species.     

Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene.

Amber, ochre and opal suppressor tRNA genes have been generated by using oligonucleotide directed site-specific mutagenesis to change one or two nucleotides in a human serine tRNA gene. The amber and ochre suppressor (Su+) tRNA genes are efficiently expressed in CV-1 cells when introduced as part of a SV40 recombinant. The expressed amber and ochre Su+ tRNAs are functional as suppressors as demonstrated by readthrough of the amber codon which terminates the NS1 gene of an influenza virus or the ochre codon which terminates the hexon gene of adenovirus, respectively. Interestingly, several attempts to obtain the equivalent virus stock of an SV40 recombinant containing the opal suppressor tRNA gene yielded virus lacking the opal suppressor tRNA gene. This suggests that expression of an efficient opal suppressor derived from a human serine tRNA gene is highly detrimental to either cellular or viral processes.     

Serine synthesis by an isolated perfused rat kidney preparation.

The isolated perfused rat kidney was shown to synthesize serine from aspartate or glutamate, both of which are also precursors of glucose. The major products of aspartate metabolism were ammonia, serine, glutamate, glucose, glutamine and CO2. Perfusion of kidneys with aspartate in the presence of amino-oxyacetate resulted in a near-complete inhibition of aspartate metabolism, illustrating the essential role of aspartate aminotransferase in the metabolism of this substrate. Radioactivity from 14C-labelled aspartate and from 14C-labelled glycerol was incorporated into serine and glucose. Production of both glucose and serine from aspartate was suppressed in the presence of 3-mercaptopicolinic acid. These data provide evidence for the operation of the phosphorylated and/or non-phosphorylated pathway for serine production to the presence of 3-mercaptopicolinic acid. This is explained by simultaneous glycolysis. The rate of glucose production, but not that of serine, was greater in kidneys perfused with glutamate or with aspartate plus glycerol than the rates obtained by perfusion with aspartate alone. These data are taken to suggest that serine synthesis occurred at a near-maximal rate, and that the capacity of the kidney for serine synthesis from glucose precursors is lower than that for glucose synthesis.     

Frameshift suppressor mutations outside the anticodon in yeast proline tRNAs containing an intervening sequence.

Extragenic suppressors of +1 frameshift mutations in proline codons map in genes encoding two major proline tRNA isoacceptors. We have shown previously that one isoacceptor encoded by the SUF2 gene (chromosome 3) contains no intervening sequence. SUF2 suppressor mutations result from the base insertion of a G within a 3'-GGA-5' anticodon, allowing the tRNA to read a 4-base code word. In this communication we describe suppressor mutations in genes encoding a second proline tRNA isoacceptor (wild-type anticodon 3'-GGU-5') that result in a novel mechanism for translation of a 4-base genetic code word. The genes that encode this isoacceptor include SUF7 (chromosome 13), SUF8 (chromosome 8), trn1 (chromosome 1), and at least two additional unmapped genes, all of which contain an intervening sequence. We show that suppressor mutations in the SUF7 and SUF8 genes result in G-to-U base substitutions at position 39 that disrupted the normal G . C base pairing in the last base pair of the anticodon stem adjacent to the anticodon loop. These anticodon stem mutations might alter the size of the anticodon loop and permit the use of a 3'-GGGU-5' sequence within the loop to read 4-base proline codons. Uncertainty regarding the exact structure of the mature suppressor tRNAs results from the possibility that anticodon stem mutations might affect sites of intervening sequence removal. The possible role of the intervening sequence in the generation of mature suppressor tRNA is discussed. Besides an analysis of suppressor tRNA genes, we have extended previous observations of the apparent relationship between tRNA genes and repetitive delta sequences found as solo elements or in association with the transposable element TY1. Hybridization studies and a computer analysis of the DNA sequence surrounding the SUF7 gene revealed two incomplete, inverted delta sequences that form a stem and loop structure located 165 base pairs from the 5' end of the tRNA gene. In addition, sequences beginning 164 base pairs from the 5' end of the trn1 gene also exhibit partial homology to delta. These observations provide further evidence for a nonrandom association between tRNA genes and delta sequences.     

Serine phosphorylation-dependent coregulation of topoisomerase I by the p14ARF tumor suppressor

p14ARF (ARF) and topoisomerase I play central roles in cancer and have recently been shown to interact. The interaction activates topoisomerase I, an important target for camptothecin-like chemotherapeutic drugs, but the regulation of the interaction is poorly understood. We have used the H358 and H23 lung cancer cell lines and purified recombinant human topoisomerase I to demonstrate that the ARF/topoisomerase I interaction is regulated by topoisomerase I serine phosphorylation, a modification that regulates topoisomerase I activity. Both cell lines express wild-type ARF and topoisomerase I proteins at equivalent levels, but H23 topoisomerase I, unlike that of H358 cells, is largely devoid of serine phosphorylation, has low activity, and complexes poorly with ARF. The ability of H23 topoisomerase I to complex with ARF can be restored by treatment with the serine kinase, casein kinase II. Consistent with these observations, we show that the response of H23 cells to camptothecin treatment is unaffected by changes in intracellular levels of ARF. However, in H358 and PC-3 cells, which express a serine phosphorylated topoisomerase I that complexes with ARF, ectopic overexpression of ARF causes sensitization to camptothecin, and siRNA-mediated down-regulation of endogenous ARF causes desensitization to camptothecin. These biological responses correlate with increased and decreased levels, respectively, of ARF/topoisomerase I complex and DNA-bound topoisomerase I. Thus, ARF is a serine phosphorylation-dependent coregulator of topoisomerase I in vivo, and it regulates cellular sensitivity to camptothecin by interacting with topoisomerase I. Certain cancer associated defects affecting ARF/topoisomerase I complex formation could contribute to cellular resistance to camptothecin.     

Nucleotide alterations in the bacteriophage T4 glutamine transfer RNA that affect ochre suppressor activity

We have determined the nucleotide sequences of the glutamine transfer RNAs that are coded by wild-type and psu₂⁺ ochre-suppressor strains of bacteriophage T4. The two transfer RNAs have the same sequence except for their anticodons, where NUG in the wild-type species is mutated to NUA in the psu₂⁺ species (N is a modified residue of U). This mutation is believed to confer suppressor activity on the psu₂⁺ glutamine tRNA. Three mutants derived from psu₂⁺ by loss of suppressor activity have been characterized with respect to their sequence alterations. Each mutant specifies a transfer RNA differing from the psu₂⁺ species by a nucleotide substitution that occupies a base-paired region in the cloverleaf arrangement of the molecule. The mutants synthesize a reduced amount of tRNA that is defective in nucleotide modifications and processing at the 5′ and 3′ termini.     

Specificity and frequency of ultraviolet-induced reversion of an iso-1-cytochrome c ochre mutant in radiation-sensitive strains of yeast

The basis for the specific pattern of ultraviolet-induced reversion of cyc1-9, an ochre allele of the structural gene for iso-1-cytochrome c, has been examined in radiation-sensitive strains of yeast. Previous analysis, using RAD⁺ strains, showed that 21 out of 23 cyc1-9 revertants induced by ultraviolet light arose by A · T to G · C transition at the first position in the UAA codon, the remaining two occurring by A · T to T · A transversion at the second position (Stewart et al., 1972; Sherman &; Stewart, 1974). All possible base-pair substitutions could be obtained with the aid of other mutagens.It has now been shown that this specificity depends largely on the action of the RAD6 locus, since ultraviolet-induced revertants of cyc1-9 arose by a variety of base-pair substitutions in a strain carrying the rad6-1 allele. Induced reversion frequencies in strains carrying this allele are much lower than normal, though significantly higher than the spontaneous frequency, and the strains are more sensitive to the lethal effects of both ultraviolet and X-irradiation. The phenotypically similar rad18-2 mutation, which appears to block the same repair pathway as rad6-1, also has some effect on the reversion specificity, but its action depends on the presence of other, unidentified, mutations. Specificity was, however, completely unaltered in an excision-defective strain carrying the rad1-2 allele. Induced reversion frequency of cyc1-9 was much higher than normal in this strain. Photoreactivation studies indicated that pyrimidine dimers were responsible for most of the revertants in RAD+, rad1 and rad6 strains. These experiments show that the RAD6+ locus is intimately concerned with error-prone repair, and suggest that excision repair is substantially error-free.