Restriction map of the Escherichia coli malA region and identification of the malT product.

A series of plaque-forming lambda h80 transducing phages carrying various portions of the malA region were isolated. A 5,800-base pair HindIII-EcoRI DNA fragment from one of these phages was cloned into pBR322 and shown to contain malT, which is the positive regulator gene of the maltose regulon, and most of malP, the structural gene for maltodextrin phosphorylase. A restriction map of the HindIII-EcoRI fragment was established, and it was correlated with the genetic map of the malA region (i) by mapping deletions which had been generated in vitro on the plasmid and (ii) by locating on the restriction map a DNA insertion of known genetic position. A 600-base pair HincII-HaeII segment was shown to contain all or part of the promoters for malT and malP, which are known to be transcribed in opposite directions. Strains carrying gene malT on a plasmid synthesized a 94,000-dalton polypeptide which was not produced by identical strains carrying similar plasmids in which malT was partially deleted. Estimates of the size of the malT gene support the conclusion that the 94,000-dalton polypeptide is the malT product.     

Suppression of amber and ochre rII mutants of bacteriophage T4 by streptomycin

Orias, E. (University of California, Santa Barbara), and T. K. Gartner. Suppression of amber and ochre rII mutants of bacteriophage T4 by streptomycin. J. Bacteriol. 91:2210-2215. 1966.-Streptomycin-induced suppression of amber and ochre rII mutants of phage T4 was studied in a streptomycin-sensitive strain of Escherichia coli and four nearly isogenic streptomycin-resistant derivatives of this strain, in the presence and in the absence of an ochre suppressor. Most of the 12 rII mutants tested were suppressed by streptomycin in the streptomycin-sensitive su(-) strain. This streptomycin-induced suppression in the su(-) strain was eliminated by the independent action of at least two of the four nonidentical mutations to streptomycin resistance. In two of the su(+)str-r strains, streptomycin markedly augmented the suppression caused by the ochre suppressor. In those su(-)str-r hosts in which significant streptomycin-induced suppression could be measured, the amber mutants were more suppressible than the ochre mutants.     

Osmoregulation of the maltose regulon in Escherichia coli.

The maltose regulon consists of four operons that direct the synthesis of proteins required for the transport and metabolism of maltose and maltodextrins. Expression of the mal genes is induced by maltose and maltodextrins and is dependent on a specific positive regulator, the MalT protein, as well as on the cyclic AMP-catabolite gene activator protein complex. In the absence of an exogenous inducer, expression of the mal regulon was greatly reduced when the osmolarity of the growth medium was high; maltose-induced expression was not affected, and malTc-dependent expression was only weakly affected. Mutants lacking MalK, a cytoplasmic membrane protein required for maltose transport, expressed the remaining mal genes at a high level, presumably because an internal inducer of the mal system accumulated; this expression was also strongly repressed at high osmolarity. The repression of mal regulon expression at high osmolarity was not caused by reduced expression of the malT, envZ, or crp gene or by changes in cellular cyclic AMP levels. In strains carrying mutations in genes encoding amylomaltase (malQ), maltodextrin phosphorylase (malP), amylase (malS), or glycogen (glg), malK mutations still led to elevated expression at low osmolarity. The repression at high osmolarity no longer occurred in malQ mutants, however, provided that glycogen was present.     

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.     

A convenient technique to compare the efficiency of promoters in Escherichia coli.

We describe a technique which allows one to insert any promoter in front of the chromosomal malPQ operon. This can be done easily by using only one plasmid, one strain, and two simple selections. Properties of the final chromosomal fusion are such that the level of amylomaltase, the product of the malQ gene, measures quantitatively the efficiency of the inserted promoter. This method was utilized to compare the efficiency of four well-known promoters: lacZp, trp, tac, lambdaPR and three malT activated promoters: malPp, malkP and malEp.     

Identification of an amber nonsense mutation in the rosy516 gene by germline transformation of an amber suppressor tRNA gene.

Seven xanthine dehydrogenase and cross-reacting material negative Drosophila melanogaster rosy stocks were screened for amber and ochre nonsense mutations. Amber and ochre nonsense suppressors were created by site-directed mutagenesis starting from a wild-type tRNA(Tyr) gene. The suppressor tRNA genes were subcloned into a pUChsneo transformation vector providing heat-shock controlled neomycin resistance. The seven rosy stocks were germline transformed with amber and ochre tDNA(Tyr), and the G1 generation was screened for Geneticin resistance. Surviving rosy516 flies transformed with the amber suppressor showed an eye colour intermediate between the original ry516 stock and the wild-type, suggesting that ry516 is an amber nonsense mutant. This was confirmed by sequencing the relevant part of the ry516 gene; the analysis revealed a C-to-T transition in a CAG glutamine codon at nucleotide 1522 of the wild-type rosy gene.     

Nonsense Motility Mutants in SALMONELLA TYPHIMURIUM

Of 313 motility-deficient mutants isolated from an LT2 his(amber) strain fixed in phase 1 by gene vh2(-), 25 regained motility when amber or ochre suppressors were introduced, in F' factors or by transduction. The fla mutants (23 amber, 1 ochre) fell in complementation groups A, B, C, F, K, a new group, M, and at least one further new group; the hypothesis of a fla gene which specifies only an RNA structural component of a flagellum-synthesizing basal apparatus is disproven for the corresponding genes. Hfr and transductional crosses confirmed gene assignments from complementation and indicated that flaM and another new fla locus map near H1. A small minority of motile bacteria were detectable in many of the amber fla mutants. In groups A and F some pairs of amber fla mutants complemented each other, and perhaps each of these groups corresponds to more than one structural gene. The suppressed derivatives of a mutant with an amber mutation in H1 made flagella morphologically and serologically indistinguishable from wild-type flagella. A slow-spreading but flagellate mutant showed mainly non-translational motility in broth, and in a viscous medium the bacteria reversed very frequently; its amber mutation, probably near H1, is inferred to cause a defect in chemotaxis, so that the bacteria give the avoidance reaction continuously.     

Site-specific mutagenesis of Escherichia coli gltT yields a weak, glutamic acid-inserting ochre suppressor

Of all the Escherichia coli tRNA genes that can give rise to an amber or an ochre suppressor by a single-nucleotide mutation, only the tRNAGlu genes have not been observed to do so. A study of the relationship between the sequences of tRNAs and the codons they translate predicts that the ochre suppressor derived from tRNAGlu would function very poorly on the ribosome. We have used site-specific mutagenesis to create the gene for such a tRNA in order to test this prediction. We cloned the tRNAGlu-Suoc gene into a high copy number plasmid, under control of the lacUV5 promoter. The mutant tRNA suppresses both amber and ochre nonsense mutations. As predicted, it is less efficient than other suppressors expressed under similar conditions.     

An amber mutation in the gene encoding the beta'' subunit of Escherichia coli RNA polymerase.

An Escherichia coli strain carrying an amber mutation (UAG) in rpoC, the gene encoding the beta prime subunit of RNA polymerase, was isolated after mutagenesis with nitrosoguanidine. The mutation was moved into an unmutagenized strain carrying the supD43,74 allele, which encodes a temperature-sensitive su1 amber suppressor, and sue alleles, which enhance the efficiency of the suppressor. In this background, beta prime is not synthesized at high temperature. Suppression of the mutation by the non-temperature-sensitive amber suppressor su1+ yields a protein which is functional at all temperatures examined (30, 37, and 42 degrees C).     

Suppression of Amber and Ochre Mutants in Salmonella typhimurium by a Mutant F′-1-gal Factor Carrying an Ochre Suppressor Gene

A Salmonella typhimurium strain was given the amber mutation hisC527 by transduction, made galactose-negative by mutation, then infected with the F'-1-gal factor. Of 107 spontaneous and mutagen-induced histidine-independent mutants tested, 3 proved to result from suppressor mutations within the F' factor. The mutant F' factors, when transferred to S. typhimurium and E. coli auxotrophs, suppressed amber and ochre but not UGA or missense mutants, and are inferred to carry ochre suppressor genes. Attempts to isolate an F' amber suppressor mutant were unsuccessful. A suppressor F' factor was transferred to 14 rough mutants which had been isolated from LT2 hisC527 (amber) by selection for resistance to phage P22.c2. One rough mutant was partly suppressed, as shown by its acquisition of O agglutinability and by alterations in its phage resistance pattern. Phage P22h grown on the suppressed mutant contransduced its rf. gene with cysE(+) and with pyrE(+), and the affected locus is inferred to be rfaL. Both the original and the mutant F' factors conferred resistance to the rough-specific phage Br60, which is therefore "female-specific."     

Mutation in the D arm enables a suppressor with a CUA anticodon to read both amber and ochre codons in Escherichia coli

Su9 of Escherichia coli differs from tRNATrp by only a G to A transition in the D arm, yet has an enhanced ability to translate UGA by an unusual C X A wobble pairing. In order to examine the effects of this mutation on translation of the complementary and wobble codons in vivo, we constructed the gene for an amber (UAG) suppressing variant of Su9, trpT179, by making the additional nucleotide change required for an amber suppressor anticodon. The resultant suppressor tRNA, Su79, is a very strong amber suppressor. Furthermore, the D arm mutation enables Su79 to suppress ochre (UAA) codons by C X A wobble pairing. These data demonstrate that the effect of the D arm mutation on wobble pairing is not restricted to a CCA anticodon. The effect extends to the CUA anticodon of Su79, thereby creating a new type of ochre suppressor. The new coding activity of Su79 cannot be explained by alterations in the level of aminoacylation, steady-state tRNA concentration, or nucleotide modification. The A24 mutation could permit unorthodox wobble pairings by generally enhancing tRNA efficiency at all codons or by altering codon specificity.     

A temperature-sensitive mutant of Escherichia coli that shows enhanced misreading of UAG/A and increased efficiency for some tRNA nonsense suppressors

Summary A spontaneous mutant was isolated that harbors a weak suppressing activity towards a UAG mutation, together with an inability to grow at 43° C in rich medium. The mutation is shown to be associated with an increased misreading of UAG at certain codon contexts and UAA. UGA, missense or frameshift mutations do not appear to be misread to a similar extent. The mutation gives an increased efficiency to several amber tRNA suppressors with-out increasing their ambiguity towards UAA. The ochre suppressors SuB and Su5 are stimulated in their reading of both UAG and UAA with preference for UAG. An opal suppressor is not affected. The effect of the mutation on the efficiency of amber and ochre suppressors is dependent on the codon context of the nonsense codon.The mutated gene (uar) has been mapped and found to be recessive both with respect to suppressor-enhancing ability as well as for temperature sensitivity. The phenotype is partly suppressed by the ochre suppressor SuC. It is suggested that uar codes for a protein, which is involved in translational termination at UAG and UAA stop codons.     

Plant nonsense suppressor tRNA(Tyr) genes are expressed at very low levels in vitro due to inefficient splicing of the intron-containing pre-tRNAs.

Oligonucleotide-directed mutagenesis was used to generate amber, ochre and opal suppressors from cloned Arabidopsis and Nicotiana tRNA(Tyr) genes. The nonsense suppressor tRNA(Tyr) genes were efficiently transcribed in HeLa and yeast nuclear extracts, however, intron excision from all mutant pre-tRNAs(Tyr) was severely impaired in the homologous wheat germ extract as well as in the yeast in vitro splicing system. The change of one nucleotide in the anticodon of suppressor pre-tRNAs leads to a distortion of the potential intron-anticodon interaction. In order to demonstrate that this caused the reduced splicing efficiency, we created a point mutation in the intron of Arabidopsis tRNA(Tyr) which affected the interaction with the wild-type anticodon. As expected, the resulting pre-tRNA was also inefficiently spliced. Another mutation in the intron, which restored the base-pairing between the amber anticodon and the intron of pre-tRNA(Tyr), resulted in an excellent substrate for wheat germ splicing endonuclease. This type of amber suppressor tRNA(Tyr) gene which yields high levels of mature tRNA(Tyr) should be useful for studying suppression in higher plants.     

UV-mutagenesis at a cloned target sequence: converted suppressor mutation is insensitive to mutation frequency decline regardless of the gene orientation

Premutational lesions produced by ultraviolet radiation in the Gln2 tRNA genes of E. coli B/r show differing sensitivities to a mutation avoidance phenomenon known as mutation frequency decline (MFD). A mutation event that changes the wild-type gene to an amber (UAG) suppressor is normally sensitive to MFD. Mutation of this amber suppressor to an ochre (UAA) suppressor is not sensitive to MFD. These two mutation events occur in the same anticodon region of the DNA. The dissimilarity of MFD sensitivity between these two mutations may result because the respective premutational photoproducts for the two are located in opposite strands of duplex DNA. To examine the effect of strand position of the premutational lesions on MFD, recombinant lambda phage were constructed that contained the amber suppressor as a mutation target in the two possible orientations. Comparison of MFD in bacterial lysogens containing either of the two types of recombinant prophage indicated that reversing the orientation of the target sequence relative to adjacent bacterial DNA had no effect on MFD. Since rotational inversion of the target sequence did not alter the sensitivity to MFD of mutation occurring at the cloned target gene, the antimutation process inherent to MFD can not be attributed to an asymmetrical interaction between the template strands and the DNA-replication complex.     

Deletion analysis of the expression of rRNA genes and associated tRNA genes carried by a lambda transducing bacteriophage.

Transducing phage lambdailv5 carries genes for rRNA's, spacer tRNA's (tRNA1 Ile and tRNA1B Ala), and two other tRNA's (TRNA1 Asp and tRNA Trp). We have isolated a mutant of lambdailv5, lambdailv5su7, which carries an amber suppressor mutation in the tRNA Trp gene. A series of deletion mutants were isolated from the lambdailv5su7 phage. Genetic and biochemical analyses of these deletion mutants have confirmed our previous conclusion (E. A. Morgan, T. Ikemura, L. Lindahl, A. M. Fallon, and M. Nomura, Cell 13:335--344, 1978) that the genes for tRNA1 Asp and tRNA Trp located at the distal end of the rRNA operon (rrnC) are cotranscribed with other rRNA genes in that operon. In addition, these deletions were used to define roughly the physical location of the promoter(s) of the rRNA operon carried by the lambdailv5su7 transducing phage.     

Polarity suppressors increase expression of the wild-type tryptophan operon of Escherichia coli

Three new polarity suppressors, selected to relieve the polar effect of nonsense mutations in the tryptophan (trp) and lactose (lac) operons of Escherichia coli, increase expression distal to nonsense mutations in both operons to a greater extent than suA. These suppressors relieve the polarity created by amber, ochre and frameshift mutations with equal efficiency.Two of the three polarity suppressors elevate enzyme synthesis in the wildtype trp operon two- and fivefold, respectively. The increase in enzyme levels is in each case correlated with increased levels and rates of synthesis of structural gene trp messenger RNA. Since expression of all genes is elevated, these findings suggest the existence of a site early in the wild-type trp operon that affects the extent of operon expression. We located the site affected by these two polarity suppressors between the operator and the first structural gene, trpE. Although the third polarity suppressor also relieves mutational polarity efficiently, it has no detectable effect on expression of the wild-type trp operon.