Genetic and enzymatic characterization of conditional lethal mutants of the yeast Schizosaccharomyces pombe with a temperature-sensitive DNA ligase.

The DNA ligase activities of wild type and temperature-sensitive lethal cdc 17 mutants of Schizosaccharomyces pombe have been studied by measuring effects on the conversion of relaxed DNA circles containing a single nick to a closed circular form. Such assays have revealed that all cdc 17 mutants have a thermosensitive DNA ligase deficiency, that this deficiency cosegregates 2:2 with their temperature-sensitive cdc-lethality in three tetrads derived from a cross against wild type, and that genetic reversion of the temperature-sensitive cdc^? phenotype is accompanied by a restoration of DNA ligase activity; all of which implies that the temperature-sensitive cdc^? phenotype of cdc 17 mutants is due to a single nuclear mutation causing a DNA ligase deficiency. Both wild type and mutant enzymes have been partially purified by chromatography in heparin/agarose columns. The wild-type enzyme is completely stable in vitro at both permissive (25 °C) and restrictive (35 °C) temperatures, whereas that of two different mutants, though completely stable at 25 °C, is rapidly inactivated at 35 °C, implying that their mutations are located in the structural gene for DNA ligase.     

Carbohydrate Accumulation and Metabolism in Escherichia coli: Characteristics of the Reversions of ctr Mutations

The reversion behavior of pleiotropic carbohydrate mutants, previously designated as ctr, was studied. The mutants revert to complete restoration of the wild-type phenotype, as well as to a spectrum of partial wild-type phenotypes. Lac⁺ reversions were found in the lac region (11 min) and some Mal⁺ reversions occurred at malB (79 min), at a distance from the site of the ctr mutations (46 to 47 min). About one-third of Lac⁺ and Mal⁺ revertants were constitutive for uptake of their respective substrates, and one-third modified for inducibility. The remaining third were not distinguishable from wild type. Induction of a ctr mutation in a lac constitutive strain, either operator or repressor mutant, did not affect lactose metabolism. A polar-like ctr mutant, deficient in both enzyme I and heat-stable protein of the phosphoenolpyruvate-dependent phosphotransferase strain was also described. Partial revertants of ctr were still found to lack enzyme I.     

Genetics of mitochondrial ribosomes of yeast: mitochondrial lethality of a double mutant carrying two mutations of the 21S ribosomal RNA gene

Summary Among the mitochondrial conditional mutations localized in the gene coding for the 21S ribosomal RNA, one — ts 902 — produces severely reduced amounts of 21S RNA and 50S subunit. We investigated its physiological properties and found that this thermosensitive mutation was associated with highly pleiotropic effects. The mutant phenotype is associated with cell death in certain conditions, and with a massive accumulation of rho^- mutants at non-permissive temperature. Furthermore, interactions with the sites of action of erythromycin and chloramphenicol, both localized within the 21S rRNA, were detected. The mutant is hypersensitive to erythromycin and has a cis-incompatibility with the chloramphenicol-resistant mutation C ₃₂₁ ^R .Ts 902 thus appears to have a dual effect, not only at the ribosomal level but also at a cellular level.     

Physiological and genetic analysis of the glucose-fructose permeation system in two Synechocystis species

Fructose is toxic for Synechocystis PCC 6714 and 6803, strains which grow chemoheterotrophically on glucose. This toxicity, as well as fructose uptake, were inhibited by glucose or by its non-metabolized analogue 3-O-methyl-glucose. The results suggested that both sugars were transported by the same permeation system, the affinity for fructose, estimated from the corresponding K _m and K _i, being very low. The unicity of the permeation system was further established by the isolation of spontaneous mutants showing the expected pleiotropic phenotype, Glu^–, Fru^r, transport^–, and by the simultaneous re-acquisition of the relevant wild type characteristics in mutant cells transformed by wild type DNA. The genetic nature of this mutation is discussed in view of the impossibility to isolate spontaneously reversed wild type clones from the transport deficient mutants.Abbreviations OMG 3-O-methyl-glucose - DCMU 3-(3,4 dichlorophenyl)-1,1-dimethylurea     

Simultaneous Transmission of Three Stable Genotypes in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

REPLICATION of the double stranded DNA genomes of bacteria takes place by a semi-conservative mechanism¹, but although autoradiographic studies have confirmed that eukaryotic DNA is replicated semi-conservatively^2,3, our lack of knowledge about the structure of the eukaryotic chromosome means that this finding does not prove that the conserved unit is a single polynucleotide strand of DNA. Indirect information supporting the hypothesis that the conserved unit consisted of a single polydeoxyribonucleotide strand has accumulated from transmission studies in chemical mutation experiments. Two phenotypic classes of mutants are readily distinguishable as a result of chemical mutagenesis; mosaic (fractional) mutants and complete (whole body) mutants. Mutation studies assume that the treated gametes contain one DNA polymer per chromosome and that the polymer is made up of two complementary nucleotide strands. With chemical mutagens (excluding acridine dyes) it is probable that only one of the two complementary strands would be chemically altered⁴. Following fertilization, DNA replication occurs, fixing both the mutational event and its complementary wild type site. The zygote will possess a mutation which will be phenotypically expressed in the adult fly depending on the early morphogenic movements in the fly's development. Assuming that only one strand is altered, the two cell lines would contain a mutant genotype and a wild type genotype. Two genotypically mutant cell lines would arise if two mutational events occur in the opposing polydeoxyribonucleotide strands within the same genie region. In this case, there would be no genotypically wild type cells in the embryo.     

Genetic and physical studies of restriction-deficient mutants of the Inc FIV plasmids R124 and R124/3

Summary R124 and R124/3 are R plasmids that carry the genes for two different restriction and modification systems. The phenotype of strains carrying either of these plasmids along with the F'lac ⁺ plasmid, is restriction-deficient (Res^-). The Res^- phenotype is not due to selection of preexisting mutants but rather to a complex mutational event caused by the F plasmid. Restriction-deficient mutants carry extensive deletions and other DNA rearrangements. Tn7 insertion is used to locate the restriction gene. Many of the Res^- mutants are genetically unstable and revert at exceptionally high frequencies. Reversion is accompanied by DNA rearrangements which result in a net gain of 9 kb of DNA. F derivates of F⁺ which do not cause restriction-deficiency but do cause deletion were used to distinguish between the DNA rearrangements associated with restriction-deficiency and those associated with deletion. From Res⁺ revertants of strains carrying F'lac ⁺ and R124 or R124/3 we have isolated F plasmids that now carry the genes for the R124 or R124/3 restriction and modification systems. It is suggested that interaction between part of the F plasmid and that segment of the R plasmid which controls the switch in Res-Mod specificity which has been observed (Glover et al. 1983) is responsible for the production of restriction-deficiency.     

Biochemical Characterization of the ctr Mutants of Escherichia coli

A test procedure based on complementation in mixed extracts is described for the assay of heat-stable protein and enzyme I of the phosphoenolpyruvate-dependent phosphotransferase system. The test was used to assay a collection of pleiotropic carbohydrate mutants of Escherichia coli (ctr mutants) and revertants of these mutants. All mutants were found to lack enzyme I of the phosphoenolpyruvate-dependent transferase system. Revertants of these mutants to complete wild phenotype regained enzyme I-forming ability. Reversion to partial wild type was not accompanied by restoration of enzyme I-forming ability.     

Pleotropic mutants from Alcaligenes eutrophus defective in the metabolism of hydrogen,nitrate, urea,and fumarate

Chromosomal mutants of Alcaligenes eutrophus unable to grow with molecular hydrogen as the energy source also failed to grow with nitrate as the terminal electron acceptor or as a nitrogen source. The mutants (Hno^–) (i) formed neither soluble nor particulate hydrogenase antigens, (ii) expressed only about 50% the wild type level of ribulosebisphosphate carboxylase activity, and (iii) transported nickel, an essential constituent of active hydrogenase, at a significantly lower rate than wild type cells. Moreover, the mutants grew very slowly with urea as nitrogen source and did not express urease. Growth on formamide was also affected and formamidase activity was induced to only a very low level. Growth of the Hno^– mutants on succinate, glutamate, fumarate, and malate was significantly slower than wild type, and a reduced rate of succinate incorporation into the mutant cells was demonstrated. The highly pleiotropic phenotype of Hno^– mutants is indicative of a chromosomal gene with a considerable physiological importance. It affected the expression of both chromosomal and megaplasmid encoded systems of energy, carbon, and nitrogen metabolism. Thus, the hno mutation restricts the metabolic versatility but does not affect the basic metabolic functions of the organism.     

Role of surface exclusion genes in lethal zygosis in Escherichia coli K12 mating

Summary We find that diaminopimelic acid in the recipient membrane is released into the medium during bacterial matings, indicating that membrane damage was inflicted on the recipient by the donor, probably for forming a channel for DNA transfer. When the damage is extensive, as in matings with an excess of Hfr bacteria, the F^- bacteria are killed (lethal zygosis). The transfer of a large amount of DNA in Hfr matings appears to enhance the killing. In analogous F⁺xF^- (Nal^r) matings, on the other hand, killing of F^- bacteria does not occur unless F plasmid transfer is inhibited by a substance like nalidixic acid. The F^- bacteria are killed, suggesting that F plasmids contain genes that express immunity to lethal zygosis in the recipient. For example, bacteria containing surface exclusion-deficient mutants of F plasmids, such as traS ^- and traT ^-, induce lethal zygosis in F^- bacteria and are susceptible to it. Various tra ^- polar mutants that abolish surface exclusion are also susceptible to lethal zygosis when mated with Hfr bacteria. Kinetic experiments indicate that in F⁺ (wild type) x F^- matings, immunity to lethal zygosis is expressed in the F^- recipient within 1/4 division time, whereas a complete expression of surface exclusion requires more than 1 division time. Thus, a complete change in all receptor sites seems to be required for the expression of surface exclusion.     

Unusual properties of a new division mutant of Escherichia coli

The properties of a division mutant of Escherichia coli were investigated. At 42 degrees C, this mutant forms nonseptate, multinucleate, filamentous cells typical of division mutants, and at the permissive temperature, is sensitive to ultraviolet (UV) irradiation. Temperature and UV sensitivities are probably due to a single mutation. The mutant phenotype is dominant to wild type. The mutant cells make DNA nearly as effectively as control cells at 42 degrees C or following UV irradiation. They exhibit normal host-cell reactivation capacities and can express all manifestations of the SOS response with the exception of Weigle reactivation. The genetic lesion which mediates this pleiotropic effect is located very close to the leu locus of the linkage map.     

Two classes of Rhizobium meliloti infection mutants differ in exopolysaccharide production and in coinoculation properties with nodulation mutants

Summary Symbiotic mutants of Rhizobium meliloti were isolated following Tn5 mutagenesis. Besides four nodulation mutants (Nod^-) unable to induce nodule formation on alfalfa, five infection mutants (Inf^-), which induce the formation of root nodules without detectable infection threads or bacteroids, were obtained. The Inf^- mutants were subdivided into two classes. One class contains mutants which fail to synthesize acidic exopolysaccharide (EPS^-). The other class is comprised of mutants which produce excess amounts of acidic exopolysaccharide (EPS^*). ¹³C nuclear magnetic resonance spectroscopy of the exopolysaccharide isolated from one of the latter type of Inf^- mutant, 101.45, revealed that the side chain of the repeating octosaccharide unit lacks the terminal pyruvate residue. Complementing cosmids were isolated for all Inf^- mutants. In the case of the Inf^- EPS^- mutants the complementing cosmids contain DNA segments which overlap and are part of megaplasmid 2. For two mutants the mutations were found to map on a 7.8 kb EcoRI fragment. In the case of the Inf^- EPS^* mutants the complementing cosmids carry chromosomal DNA. The mutations of two Inf^- EPS^* mutants were localized on a 6.4 kb EcoRI fragment. Coinoculation of alfalfa plants with Nod^- and Inf^- EPS^- mutants resulted in effective symbiosis. The nodules appeared wild type and fixed nitrogen. In constrast, coinoculations with Nod^- mutants and the Inf^- EPS^* mutant 101.45 did not result in the formation of effective nodules.     

Cytoplasmically determined human cell mutants defective in mitochondrial ribosome assembly

Summary The mitochondrial macromolecular synthesis and assembly processes of three mutants of the human cell line VA₂-B, which are deficient to varying degrees in mitochondrial protein synthesis and resistant to chloramphenicol (CAP), have been analyzed. The mutant VA₂/CAP23 was selected directly for resistance in vivo to CAP, while the mutants VA₂/mtPS^- 1 and 4 were selected as respiration deficient, and subsequently found to be resistant to CAP. The phenotypes of the three mutants are inherited cytoplasmically and thus the mutations are probably localized in mitochonrial DNA (mtDNA). The gross mtDNA sequence organization of the three mutants, as analyzed by digestion with several restriction enzymes, was found to be indistinguishable from that of VA₂-B cells, and their relative mtDNA content comparable to or greater than that of the parental cell line. No difference was observed in the electrophoretic mobilities of mitochondrial rRNAs or mRNAs of the mutants analyzed on denaturing methylmercuric hydroxide-agarose gels as compared to the mobilities of the RNA species of the VA₂-B cells. Similarly, no significant change, or only a moderate decrease in the rate of synthesis of mitochondrial 12s or 16s rRNAs was found in the mutants. By contrast, the incorporation of³H-uridine into mitochondrial ribosomal subunits was significantly reduced in the three mutants, and in each of them, this decrease paralleled the decrease in the level of mitochondrial protein synthesis. The complex phenotypes of the mutants analyzed here could be accounted for by assuming that, as previously reported for other CAP-resistant mouse and human cell variants, the 16s rRNA gene is the site of the mutation. Such a mutation would produce a CAP-resistant phenotype and at the same time affect the assembly of the large ribosomal subunit, with a resulting reduction in mitochondrial protein synthesis and a pleiotropic respiratory deficiency. However, other possible sites for the primary defect in the mutants analyzed here cannot be excluded.     

Plasmid-mediated control of nodulation in Rhizobium trifolii

The nodulation ability was effectively eliminated from different Rhizobium trifolii strains incubated at elevated temperature (urkowski and Lorkiewicz, 1978). Non-nodulating (Nod^-) mutants were stable and no reversion of Nod^- to Nod⁺ phenotype was observed. Strains R. trifolii 24 and T12 which showed a high percentage of elimination of nodulation ability were examined in detail. Two plasmids were detected in strain 24 using neutral and alkaline sucrose gradient centrifugation of plasmid preparations. Molecular weights of the plasmids pWZ1 and pWZ2 were 460 Mdal and 190 Mdal, respectively. Rhizobium lysates labeled with ³H-thymidine and ultracentrifuged in caesium chloride — ethidium bromide gradients demonstrated a 40% reduction of the plasmid DNA content in R. trifolii 24 Nod^- mutants in comparison with the nodulating wild type strain 24. It was found further that non-nodulation of mutants 24 Nod^- was due to the absence of plasmid pWZ2. Sucrose gradient data also demonstrated that strain T12 contained two plasmids with molecular weights corresponding to those of pWZ1 and pWZ2, respectively. In Nod^- mutant clones derived from strain T12, pWZ2 plasmid was missing.Non Standard Abbreviations CCC covalently closed circular - OC open cirucular - Sarkosyl sodium N-lauroylsarcosinate     

Abolition of the cyclic variations in radiosensitivity during meiosis in a sporulation mutant blocked in premeiotic DNA synthesis

Summary The response to ultraviolet light (254 nm) of two sporulation mutants during the meiotic process was compared to that of a wild type diploid strain of Saccharomyces cerevisiae. The cyclic pattern for cell killing and rho ^- induction characteristic of diploid wild type cells persists in a strain able to perform the premeiotic DNA synthesis but which is blocked in the further steps of meiosis (spo8 DMS1). On the contrary, these fluctations are abolished in a derived mutant (spo8 dsm1) which is blocked in the premeiotic DNA synthesis. Under these conditions, the response to cell killing can be dissociated from that observed for rho ^- induction.     

A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair.

The saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase delta holoenzyme activity was achieved at 37 degrees C but not at 14 degrees C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase delta but completely defective for interaction with DNA polymerase epsilon. Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication.     

Comparison of RNA's transcribed in vivo from mitochondrial DNA of cytoplasmic and chromosomal respiratory deficient mutants

Summary In some respiratory deficient cytoplasmic mutants, the buoyant density of mitochondrial DNA is changed to detectable degrees, as compared to that of wild type strain: since this density shift suggests an important modification of polynucleotide sequence in mitochondrial DNA, we examined sequence homology between mitochondrial DNA of the respiratory mutants issued from cytoplasmic or chromosomal mutations. Mitochondrial DNA, nuclear DNA and total RNA were extracted from ϱ⁺ cells (wild type, respiratory sufficient) and from ϱ^- cells (cytoplasmic “petite colonie” mutant, respiratory deficient), and molecular hybridization experiments were carried out between them. When ϱ⁺ RNA × ϱ⁺ mitochondrial DNA, formed roughly twice as much hybrids as the heterologous cross, ϱ⁺ RNA × ϱ¹ mitochondrial DNA. Reciprocally, when ϱ^- RNA was hybridized to ϱ⁺ and ϱ^- mitochondrial DNA, the homologous cross produced again about twice as much hybrids as the heterologous cross. These results were confirmed by dehydridization-rehybridization experiments: the RNA separated from the hybrids “ϱ⁺ RNA × ϱ⁺ mit-DNA” as well as the RNA separated from the hybrids “ϱ⁺ RNA × ϱ^- mit-DNA” were rehybridized either with ϱ⁺ or ϱ^- mit-DNA. A preferential hybridization of ϱ⁺ RNA with ϱ⁺ mit-DNA, and of ϱ^- RNA with ϱ^- mit-DNA was clearly observed. On the contrary, ϱ⁺ and ϱ^- nuclear DNA did not distinguish ϱ⁺ or ϱ^- RNA. The same series of experiments were carried out using a chromosomal mutation,P ₇ to p₇, leading to the same respiratory deficient phenotype. We found that the p₇ mutation did not introduce a detectable change in mitochondrial DNA base sequence. The results support the idea that the cytoplasmic hereditary factor, ϱ, resides in mitochondrial DNA and that the ϱ^- mutations studied correspond to a dispersed sequence modification covering about a half of the total mitochondrial DNA genome, leaving the other half unchanged. Alternatively, the results can be explained by a hypothesis in which mitochondrial DNA is a heterogeneous population of the molecules having more or less related sequences and the mutation leads to a selection of certain molecular species. 4 S RNA was found to contain RNA species which hybridize with mitochondrial DNA. The degree of hybridization was very different for ϱ⁺ and ϱ^- S RNA, when they were hybridized with either ϱ⁺ or ϱ^- mitochondrial DNA.     

A mutation in the <Emphasis Type="Italic">Cap Binding Protein 20</Emphasis> gene confers drought

In a genetic screen for Arabidopsismutants displaying pleiotropic alterations in vegetative development and stress responses we have identified a T-DNA insertion mutation in the Cap Binding Protein 20 (CBP20) gene, that encodes the 20kDa subunit of the nuclear mRNA cap binding complex (nCBC). Plants homozygous for the recessive cbp20 mutation show mild developmental abnormalities, such as serrated rosette leaves, delayed development and slightly reduced stature. Loss of the cbp20 function also confers hypersensitivity to abscisic acid during germination, significant reduction of stomatal conductance and greatly enhanced tolerance to drought. Expression of the wild type cDNA by CaMV35S promoter provides full genetic complementation of the pleiotropic cbp20phenotype. Phenotypic characteristics of the cbp20 mutant are very similar to those of recently described abh1mutant that is defective in the 80kDa subunit of nCBC. Our data thus confirm that both genes are dedicated to the same function. CBP20 provides a new target for breeding efforts that aim at the improvement of drought tolerance in plants. Our results also show that screening for pleiotropic phenotypes in mutant plant populations may be a fruitful strategy to isolate genes for agronomically important traits.     

Three <Emphasis Type="Italic">nth</Emphasis> homologs are all required for efficient repair of spontaneous DNA damage in <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>

Deinococcus radiodurans is a bacterium that can survive extreme DNA damage. To understand the role of endonuclease III (Nth) in oxidative repair and mutagenesis, we constructed nth single, double and triple mutants. The nth mutants showed no significant difference with wild type in both IR resistance and H₂O₂ resistance. We characterized these strains with regard to mutation rates and mutation spectrum using the rpoB/Rif^r system. The Rif^r frequency of mutant MK1 (△dr0289) was twofold higher than that of wild type. The triple mutant of nth (ME3)generated a mutation frequency 34.4-fold, and a mutation rate 13.8-fold higher than the wild type. All strains demonstrated specific mutational hotspots. Each single mutant had higher spontaneous mutation frequency than wild type at base substitution (G:C → A:T). The mutational response was further increased in the double and triple mutants. The higher mutation rate and mutational response in ME3 suggested that the three nth homologs had non-overlapped and overlapped substrate spectrum in endogenous oxidative DNA repair.     

DNA sequence analysis of point mutations in traA, the F pilin gene, reveal two domains involved in F-specific bacteriophage attachment

Summary Six missense point mutations in traA (WPFL43,44,45,46,47 and 51), the gene encoding F pilin in the transfer region of the F plasmid, have been characterized for their effect on the transfer ability, bacteriophage (R17, QB and fl) sensitivity and levels of piliation expressed by the plasmid. The sequence analysis of the first five of these mutations revealed two domains in the F pilin subunit exposed on the surface of the F pilus which mediate phage attachment. These two domains include residues 14–17 (approximately) and the last few residues at the carboxy-terminus of the pilin protein. One of these mutants had a pleiotropic affect on pilus function and was thought to have affected pilus assembly. The sixthe point mutant (WPFL51), previously thought to be in traA, was complemented by chimeric plasmids carrying the traG gene of the F transfer region, which may be involved in the acetylation of the pilin subunit. A traA nonsense mutant (JCFL1) carried an amber mutation near the amino-terminus which is well suppressed in SuI⁺ (supD) and SuIII⁺ (supF) strains. Neither the antigenicity of the pilin nor the efficiency of plating of F-specific bacteriophages were affected when this plasmid was harbored by either suppressor strain. A second amber mutant (JCFL25) which is not suppressible, carried its mutation in the codon for the single tryptophan in F pilin, suggesting that this residue is important in subunit interactions during pilus assembly. Two other point mutants (JCFL32 and 44) carried missense mutations in the leader sequence (positions 9 and 13) which affected the number of pili per cell presumably by altering the processing of propilin to pilin.     

Characterization of an amber suppressor in Pneumococcus.

Summary Partial revertant has been isolated, with resistance to aminopretin intermediate between wild type and mutant. This phenotype is the result of a mutation at a gene unlinked to the amiA locus. This suppressor mutation (su⁺) has no phenotypic characteristics by itself except a slow growth. 9 amiA mutants (belonging to 6 sites) are affected by su⁺ out of the 30 investigated mutants (i.e. 22 sites). The efficiency of suppression is site dependent. Two sites out of 14 mutants belonging to the thymidilate synthetase gene are suppressible. Thymidilate synthetase activity is partially restored by su⁺. Optochin mutants can also be suppressed. Thus su⁺ is not gene specific but site specific. Moreover when the str-41 allele conferring resistance to streptomycine is introduced by transformation, the suppression effect is restricted. All these properties are characteristic of an informational suppressor.The t-RNA extracted from the suppressor strain su⁺ but not the wild type restored the synthesis of coat protein coded by RNA from an amber mutant of bacteriophage f2. Attempts to detect ochre suppression activity gave negative results. It is suggested that the su⁺ gene is amber specific.Thus su⁺ can provide insight into the nature of suppressible mutations which should be point mutations. Both low efficiency and high efficiency mutants are affected by su⁺; this is additional evidence that both categories contain point mutations.