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.     

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."     

Process of Infection with Bacteriophage φX174: XXXV. Cistron VIII

Twenty-two new amber and ochre mutants of phiX174 were isolated and classified into complementation groups. Three ochre mutants gave positive complementation tests with reference mutants in the seven previously defined groups and thus represent an eighth cistron. Studies of the physiology of infection in the nonpermissive condition for mutants in cistron VIII yielded the following information. (i) Replicative-form synthesis proceeds at a normal rate, and is turned off at the usual time. (ii) Synthesis of single-stranded deoxyribonucleic acid (DNA) is delayed until nearly 40 min after infection (in the absence of lysis), at which time a slow synthesis of infectious phage particles commences. The synthesis of infectious particles at late times is interpreted as a consequence of "leakage," and indicates that the cistron VIII product is required in very small quantities. (iii) During the normal period of single-strand synthesis, most of the replicative-form DNA is found in a form with properties similar to those of the transient intermediates of single-strand DNA synthesized during normal infection.     

Histidinol Dehydrogenase (hisD) Mutants of Salmonella typhimurium

A multidisciplinary analysis has been applied to over 150 hisD mutants of Salmonella typhimurium in a study of gene-enzyme relationship. The mutants were examined for production of immunologically cross-reacting material by using antibody to purified histidinol dehydrogenase, and for genetic complementation by using a set of F' factors bearing Escherichia coli hisD complementing mutants. Classifications as to missense, nonsense, frameshift, or deletion mutant are proposed on the basis of mutagenesis and suppression tests. For the suppression tests the mutants were examined both by a simultaneous suppression technique and by testing for response to E. coli F' factors bearing a recessive lethal amber and a recessive lethal ochre suppressor. The data are interpreted in relation to the position of the mutations in the recombination and complementation maps and in relation to the known composition of histidinol dehydrogenase. The gene hisD appears to be single cistron for the production of a single biosynthetic polypeptide.     

Variation of Mutagenic Action on Nonsense Mutants at Different Sites in the Iso-1-Cytochrome c Gene of Yeast

Three ochre and two amber mutants in yeast have been definitively identified by the amino acid replacements in iso-1-cytochromes c from intragenic revertants. Except for rare and sometimes unusual changes, all of the replacements were single amino acids whose codons differed from UAA or UAG by one base. These assignments, which were based on the absence of tryptophan replacements in ochre revertants, could be corroborated from the studies of two groups of suppressors that were shown to act on either the ochre or amber mutants. All five nonsense mutants are located at different sites in the cyc1 gene and all are at sites that can be occupied by amino acids having a wide range of structures. The relative frequencies of the amino acid replacements indicate that identical codons located at different sites may respond differently to a mutagenic agent. Notably glutamine replacements occurred almost exclusively in UV-induced revertants of only one ochre mutant cyc1–9, but not at all or at reduced proportions in the others. Similarly, lysine replacements occurred almost exclusively in the NA-induced revertants of only the ochre mutant cyc1–72, but not at all in the others. These and other results reveal that mutation of A·T base pairs by UV and nitrous acid are dependent upon the location of the codon within the gene as well as the location of the base pair within the codon. From these findings, it appears as if the type of base-pair changes induced by UV and nitrous acid are strongly influenced by adjacent nucleotide sequences.     

Genetic Analysis of Hybrid Strains Trisomic for the Chromosome Containing a Fatty Acid Synthetase Gene Complex (fas1) in Yeast

Evidence of spontaneous n+1 aneuploidy has been obtained by trisomic segregation analysis of four independently maintained stocks of Saccharomyces cerevisiae defective in saturated fatty acid synthesis (fas1). In all cases tested, only the chromosome bearing the mutant fatty acid locus was disomic. Tetrad analysis of trisomic hybrids enabled the identification of chromosome XI as the one bearing the fatty acid locus and the assignment of fragment 5 to chromosome XI. Statistical analysis of tetrad frequencies generated by markers in triplex configuration provided information on the meiotic configuration of pairing of the three homologous chromosomes. The possible relationship between defective nuclear membranes and the disjunction of chromosomes in fas1 strains is discussed.     

A Genetic Characterization of the Nadc Gene of Salmonella Typhimurium

The nadC gene of Salmonella encodes the pyridine biosynthetic enzyme PRPP-quinolinate phosphoribosyltransferase. Using a combination of genetic techniques, a deletion map for the Salmonella nadC gene has been generated which includes over 100 point mutants and 18 deletion intervals. The nadC alleles obtained by hydroxylamine mutagenesis include those suppressed by either amber, ochre, or UGA nonsense suppressors as well as alleles suppressed by the missense suppressor, sumA. Deletions were obtained by three separate protocols including spontaneous selection for loss of the nearby aroP gene, recombination between aroP::MudA and nadC::MudA insertion alleles, and selection for spontaneous loss of tetracycline resistance in a nearby guaC::Tn10dTc insertion mutant allele. The nadC mutants comprise one complementation group and the nadC+ allele is dominant to simple, nadC auxotrophic mutant alleles. Intragenic complementation of two nadC alleles, nadC493 and nadC494, mapping to deletion intervals 17 and 18, respectively, suggests that nadC encodes a multimeric enzyme. Both nadC and the nearby aroP locus are transcribed counterclockwise on the standard genetic map of Salmonella, in opposite orientation to the direction of chromosome replication.     

The CAN1 Locus of SACCHAROMYCES CEREVISIAE: Fine-Structure Analysis and Forward Mutation Rates

A system of strains and growth media was developed to allow efficient detection of forward mutation, reversion, complementation, and suppression at the canavanine-resistance (CAN1) locus of Saccharomyces cerevisiae. Genetic fine-structure analysis revealed that the map length is at least 40, and possibly as much as 60 X-ray map units; this is the longest gene map yet reported in S. cerevisiae. Allelic complementation was not observed, despite testing of a large number of allele pairs, and alleles suppressible by the ochre suppressor SUP11 were absent from a sample of 48 spontaneous mutants and occurred infrequently (7%) among a sample of ultraviolet-induced mutants. Infrequent mutant types included canavanine-resistant mutants capable of arginine uptake and alleles thought to represent deletions or inversions. In contrast to previous reports in the literature, the spontaneous forward mutation rate at CAN1 did not increase during meiosis.     

Conjugational complementation analysis of transfer-deficient mutants of Flac in Escherichia coli

A series of 102 transfer-deficient (tra(-)) mutants of Flac (84 of which have been previously described) were classified as carrying frameshift, amber, ochre, UGA, and nonsuppressible mutations. Techniques were evolved for using cultures of F prime strains as efficient recipients in matings, for measuring the number of (F'/F') heterozygote cells in transient populations, and for measuring complementation between different Flac tra(-) mutants. These techniques were used to define nine tra cistrons and to assign most of the tra(-) mutations to one or other of these.     

Specific induction of transitions and transversions of G-C base pairs by 4-nitroquinoline-1-oxide in iso-1-cytochrome c mutants of yeast

The base-pair changes induced by the highly carcinogenic agent, 4-nitroquinoline-1-oxide, have been determined from the reversion rates of defined tester strains and from the amino acid replacements of revertant iso-1-cytochromes c. The mutant codons and the base-pair changes of reverse mutations of 14 cyc1 mutants were previously determined from alterations of iso-1-cytochromes c in intragenic revertants. These 14 cyc1 mutants, which were used as tester strains, included nine mutants with altered AUG initiation codons, an ochre (UAA) mutant, an amber (UAG) mutant and three frameshift mutants (Stewart et al., 1971,1972; Stewart &; Sherman, 1972,1974; Sherman &; Stewart, 1973). NQO² induced a high rate of reversion in the initiation mutant cyc1-131, the only mutant in the group which reverts to normal iso-1-cytochrome c by a G · C → A · T transition. In addition, NQO produces a significant rate of reversion of all cyc1 mutants which revert by G · C transversions, e.g. the amber (UAG) mutant and the initiation mutants containing AGG, and probably CUG mutant codons. It did not revert the ochre mutant which contains no G · C base pairs. Ten NQO-induced revertants of the amber mutant cyc1-179 contained the expected replacements of residues of tyrosine, and ten NQO-induced revertants of each of the cyc1-131 and cyc1-133 initiation mutants all contained the expected normal iso-1-cytochrome c. The structures of these iso-1-cytochromes c and the pattern of reversion of the tester strains indicate that base-pair substitutions arise at G · C base pairs which are the site of NQO attack. Thus NQO induces G · C → A · T transitions, G · C → T · A transversions and possibly G · C → C · G transversions. Because of its mode of action, NQO may be useful in less-defined systems for identifying G · C base pairs in mutant codons.     

Deficiency of double mutant recombinants in crosses of phage T4

Summary Only 1.4% of the double mutant recombinants expected on the basis of wild-type recombination frequencies were observed in the combined data from two-factor crosses between a gene 37 amber mutant, amB280, and eighteen different temperature sensitive mutants which were also defective in gene 37. Similar, though less extreme, deficiencies of double mutant recombinants were observed by Doermann and Parma (1968) for mutants in several other genes. In our amB280xts crosses, frequencies of wild-type recombinants were in reasonably good agreement with those expected from the map positions of the mutants determined in crosses not involving amB280. Wild-type and double mutant recombinants were found at comparable frequencies when each of three other gene 37 amber mutants was crossed to a gene 37 temperature sensitive mutant.Experiments were performed to test whether the deficiency of double mutant recombinants in the amB280xts crosses could be explained by assuming that they occurred primarily in heterozygous particles, where their expression was masked. However, no evidence in support of this explanation was found. Other possible explanations, that the deficiency of double mutants was due to their inviability or the inability of double mutant chromosomes to replicate, were also inconsistent with our observations. The hypothesis considered to most plausibly explain our evidence is that the process by which double mutant recombinant chromosomes are formed is inhibited in the vicinity of a poorly suppressed am mutation.     

Nonsense Mutants in the rII A Cistron of Bacteriophage T4

After in vitro treatment of bacteriophage T4 with hydroxylamine (HA), 54 nonsense mutants in the rII A cistron were isolated. These mutants were characterized by growth on suppressor strains of Escherichia coli, and the mutational sites were mapped in the rII A cistron. Twenty-five (9 sites) were amber (UAG), 20 (6 sites) were opal (UGA), and 9 (6 sites) were ochre (UAA). Mapping experiments further indicated that there were three closely linked pairs of amber and opal mutations, conceivably involving mutations occurring in adjacent nucleotides. Based on the specificity of HA mutagenesis (GC → AT), the amino acid codons in which the mutations occurred have been inferred. It is suggested that the three amber-opal pairs arose in tryptophan codons (UGG) and the six ochre mutants arose in glutamine codons (CAA). The six unpaired ambers and the three unpaired opals have been tentatively assigned to glutamine codons (CAG) and arginine codons (CGA), respectively, in the wild-type phage.     

Differentiation between Amber and Ochre Mutants of Yeast by Reversion with 4-Nitroquinoline-1-Oxide

4-nitroquinoline-1-oxide (NQO) induces high frequencies of intragenic revertants of amber (UAG) but not ochre (UAA) mutants of yeast. Distinction of the amber and ochre codons was made with well-characterized nonsense mutants of the iso-1-cytochrome c gene (cyc1 mutants) as well as with nonsense mutants having nutritional requirements. Thus the NQO-induced reversion frequencies corroborated the assignments that were based on the pattern of amino acid replacements in intragenic revertants and on the speficity of suppression. It was concluded from these results and from the results of a previous investigation with other cyc1 mutants (Prakash, Stewart and Sherman 1974) that NQO induces transversions of G:C base pairs at many sites and that the specificity is not strongly influenced by neighboring base pairs in at least the strains examined in these studies. NQO was previously shown to induce G:C → A:T transitions at least at one site and this and the previous study established that it does not significantly mutate A:T base pairs at numerous sites. Thus NQO can be used to selectively mutate G:C base pairs and to determine if the pathways of reverse mutations involve G:C base pairs. Suppressors that act on either amber or ochre mutants were induced with NQO, indicating that they can arise by mutations of G:C base pairs.     

Genetic and Physiological Characterization of met15 Mutants of SACCHAROMYCES CEREVISIAE: a Selective System for Forward and Reverse Mutations

One hundred and thirty-three spontaneous and induced mutants of the met15 locus in Saccharomyces cerevisiae were characterized with respect to temperature sensitivity, osmotic remediability, interallelic complementation, and suppressibility by amber and ochre suppressors. Forty mutants are osmotic remedial; 17 of these, and no others, are also temperature-sensitive. Seven of 133 mutations are suppressible by an amber suppressor and 11 are suppressible by an ochre suppressor. Seventy percent of the mutants exhibited interallelic complementation, suggesting that the functional gene product of the met15 gene is a multimeric protein. Relative map positions of 30 met15 were estimated from the frequencies of X-ray-induced mitotic reversion of various heteroallelic diploids. All complementing nonsense mutations are located near one end of the gene in contrast to other nonsense mutations which span most of the gene, thus relating the direction of translation of the mRNA with respect to the fine-structure map. Recombination studies indicated that two of 30 mutants contained deletions of the entire met15 locus.—It was established that a variety of mutational types, including missense, nonsense, and deletions, are recovered with this unique system in which both forward and reverse mutations can be selected on the basis of methyl mercury resistance and methionine requirement of the met15 mutants.     

A new system for studying molecular mechanisms of mutation by carcinogens.

A new system for studying the molecular mechanisms of mutation by carcinogens is described. The system involves (a) site-specific modification of the essential gene G in phi X174 replicative form DNA by a combination of chemical and enzymatic steps; (b) production of mutant virus carrying a change at a single preselected site by transfection of spheroplasts with the site modified phi X174 DNA; (c) detection and propagation of mutants using a host carrying the plasmid, p phi XG, that rescues all type of gene G mutants by complementation; (d) identification of the mutation in the progeny virus by isolating and sequencing mutant phi X174 DNA in the region that carried the parental, site-specific change. To demonstrate that this system is operational, we have produced a previously unknown phi X174 gene G mutant carrying a C leads to T base change at position 2401 of the viral (plus) strand. This preplanned, nonsense (amber) mutant was obtained by changing G to A at the appropriate position in a chemically synthesized, octadeoxynucleotide, minus strand primer; elongating this enzymatically with Escherichia coli DNA polymerase I (larger fragment) (lacking 5' leads to 3' exonuclease activity) to a 17-mer; and repriming to obtain the site-modified phi X174 replicative form DNA enzymatically with E. coli DNA polymerase I (large fragment) and T4 DNA ligase. After transfection of spheroplasts with the heteroduplex DNA, the lysate was screened for mutant virus with permissive (carrying p phi XG) and nonpermissive (without p phi XG) host cells. About 1% of the progeny virus were mutants. Out of 15 isolates, 11 were suppressible by an amber Su1+ (serine) or an ochre Su8+ (glutamine) suppressor. The other 4 isolates were not suppressed at all. Replicative form DNA produced from one of the suppressible mutants was shown (by sequencing) to contain the expected C leads to T change at the preselected site in the viral strand. Replicative form DNA from one of the nonsuppressible mutants was partially sequenced. No change was found at or around position 2401. The nature of the mutation(s) in these isolates is still unknown. The occurrence of mutations outside the preselected sites represent a potential problem for our projected studies, but additional data is required before the problem can be fully evaluated. In spite of this, it should be possible to study, in vivo, the biological effects of any site-specific modification (including covalent modifications by carcinogens) that can be introduced into gene G of phi X174 DNA via a synthetic, oligonucleotide primer.     

Marker Effects in the Genetic Transduction of Tryptophan Mutants of E. COLI

Recombination frequencies have been determined in crosses involving 28 mutant strains for 20 of which the site of the alteration is known from studies of amino-acid substitutions in the protein products. Three of these mutants showed especially high frequencies of recombination when crossed to other single mutants or when crossed to a strain carrying two alterations at opposite ends of the trpA gene. There is no obvious molecular explanation of the high recombination of these three mutants. They include one missense mutant, one amber and one ochre. The low-frequency recombination mutants include all these same classes as well as frameshift mutants. There is nothing unique about the intragenic location of the high-recombination mutants; in each case there is at least one low-recombination mutant in the same codon.-Crosses involving mutants which were isolated in an altered wild type have shown that the behavior of a high-recombination mutant does not result from its molecular configuration alone, but from its combination with the homologous wild-type sequence from the other parent.-Several lines of evidence indicate that recombination in this system frequently involves closely-spaced double exchanges (about 40 codons apart).     

Blue ghosts: a new method for isolating amber mutants defective in essential genes of Escherichia coli.

We describe a technique which permits an easy screening for amber mutants defective in essential genes of Escherichia coli. Using this approach, we have isolated three amber mutants defective in the rho gene. An extension of the technique allows the detection of ochre mutants and transposon insertions in essential genes.     

Characterization of amber and ochre suppressors in Salmonella typhimurium.

Amber and ochre suppressor mutations in Salmonella typhimurium were selected. The amino acid insertions directed by the suppressors were inferred from suppression patterns of Escherichia coli lacI amber mutations. These amber mutations only respond to nonsense suppressors that direct the insertion of particular amino acids. Four Salmonella amber suppressors characterized insert serine, glutamine, tyrosine, and (probably) leucine. Of the three ochre suppressors characterized, two direct the insertion of tyrosine and one directs that of lysine. Of the three amber and two ochre suppressors which have been mapped by phage P22 cotransduction, all are located in the same relative position on the Salmonella map as the analogous E. coli suppressors are on the E. coli map.