Reversion of the gal3 mutation of Escherichia coli: Partial deletion of the insertion sequence

Summary The gal3 mutation of E. coli is an insertion of a DNA sequence, 1,100 base pairs in length, into the operator-promoter region of the galactose operon. This mutation reverts spontaneously to gal⁺ by excision of the insertion to produce stable, inducible revertants, or by tandem duplications of the gal operon to produce unstable, constitutive revertants. The nature of a third class of revertants, which are stable and constitutive, is the subject of the present study.The stable, constitutive class of revertants included approximately 30% of all gal⁺ revertants obtained from a gal3() strain. Although the constitutive reversions could be transduced by , the efficiency was found to be extremely poor and the rare transductants which did appear seemed to originate from abnormal transducing particles. It was concluded that these reversions were not normally packaged by .In order to facilitate the packaging of these reversions, the chlD-pgl region was deleted from the parent gal3() strain. Unexpectedly, the gal3 mutation in the majority of these deletions reverted to produce stable, constitutive reversions exclusively. The explanation proposed was that the chlD-pgl deletions had also removed part of the gal operator-promoter up to the gal3 insertion, so that simple excisions of the insertion yielded stable, constitutive revertants by connecting the gal structural genes to a different promoter. These revertants were not considered to be true representatives of the stable, constitutive class. The specificity of deletion end-points at the insertion was found only in the gal3() strain, and not in gal ⁺, gal ⁺(), or gal3 strains. Moreover, the frequency of spontaneous chlD-pgl deletions increased 10- to 15-fold in presence of the gal3 insertion.A gal phage bearing a true stable, constitutive reversion (gal ^c 200) was isolated from the revertant strain by subsequent deletion of the chlD-pgl segment (31). Electron micrographs of gal ⁺ and gal ^c 200 31(chlD pgl) DNA heteroduplexes were interpreted to indicate that the stable, constitutive reversion had arisen by a deletion of 3/4 of the gal3 insertion sequence.The main conclusions are: (i) the stable, constitutive reversions of gal3 can arise by partial deletions of the insertion sequence, apparently by elimination of the nucleotide sequence which causes polarity; (ii) the chlD-pgl deletions may exhibit preferential termination at the right extremity of the gal3 insertion in presence of prophage ; and (iii) the gal3 insertion appears to inhibit the production of gal particles by providing a nucleotide sequence which is recognized and degraded by a specific endonuclease. It is suggested that inhibition of transducing particle formation by gal3 and the preferred termination of deletions at gal3 might represent related phenomena.     

Tandem duplications of the histidine operon observed following generalized transduction in Salmonella typhimurium

Unstable merodiploid transductants may be observed among the progeny of certain generalized transductional crosses between complementing mutations in the histidine operon of Salmonella typhimurium. In the presence of a functional recombination system, these transductants are unstable and they segregate His^? clones of both parental genotypes. The properties of these His⁺ transductants suggest that they contain tandem duplications of a region of DNA which includes the histidine operon, such that each copy of the duplication contains one of the two complementing mutations involved in the transduction. Transductional duplications have been observed from 14 pairs of his mutations, but only with complementing pairs of parental mutations. The length of duplicated material may be quite large: two duplications were found to include genetic markers ten minutes removed from the histidine operon on the Salmonella chromosomal map.These transductants appear to arise in a subpopulation of recipient cells which contain pre-existing tandem duplications of the histidine operon. As much as 0.01 to 0.1% of the cell population appears to be tandemly duplicated for a chromosomal region which includes the histidine operon.     

Nature of deletions formed in response to IS2 in a revertant of the gal3 insertion of E. coli.

Summary The gal3 mutation of E. coli, which arose by the insertion of IS2 in the OP region of the gal operon, reverts spontaneously by excision of the IS2 to produce inducible revertants or by mutational alterations of IS2 to produce constitutive revertants. However, gal3() strains bearing chlD-pgl deletions produce constitutive revertants alone. We proposed that deletions formed in the presence of IS2 terminate specifically at its right end, so that revertants arising by excision of IS2 fuse the gal genes to other promoters. Therefore, the revertants are exclusively constitutive.The above hypothesis was tested by electron microscopy of IS2-specific deletions. Spontaneous chlD-pgl deletions were isolated from gal ^c331 (a revertant of gal3 which retains IS2) and transferred to gal genomes. Electron microscopy of DNA heteroduplexes from these phages confirmed that all of the deletions examined have one end-point fixed at the right end of IS2, whereas their other end-points are variable. In each case, the complete IS2 element was apparently retained. This specificity was also detectable in a revertant (gal ^c200) which retains only the right 1/5 portion of the IS2. The frequencies of these deletions were generally increased in constitutive revertants of gal3. Since a galO ^cmutant did not show a similar increase, it seems that this effect depends upon a base sequence provided by IS2. Moreover, the presence of prophage contributes to the specificity and, in some instances, the frequency of IS2-specific deletions.A mechanism for the formation of the IS2-specific deletions has been proposed. A base sequence located at, or near, the right end of IS2 is recognized and nicked by a specific endonuclease. The nick is enlarged by unidirectional, exonucleolytic degradation to produce deletions extending outwards from the insertion. In constitutive revertants, the nicking site may be exposed to endonucleolytic attack more frequently.     

Leakiness of Pleiotropic Maltose-negative, Bacteriophage λ-resistant Mutants of Escherichia coli K-12

Cultures of Escherichia coli K-12 malA(-)lambda(r)gal(-) can be transduced to gal(+) by bacteriophage lambdadg because of leakiness of the lambda(r) phenotype. The efficiency of such transduction is about 10(-5) that of transduction of mal(+)lambda(s) bacteria. Leaky cells (lambda(s)phenocopies) adsorb only very few phage particles, and many transductants, therefore, are defective heterogenotes or show integration of the gal(+) gene, which is unaccompanied by lysogenization.     

Isolation of Transducing Bacteriophages for the Histidine and Isoleucine-Valine Operons in Escherichia coli K-12

In vitro studies have been of great value in elucidating the mechanism of the regulation of several bacterial operons. To obtain a deoxyribonucleic acid preparation enriched for the histidine (his) and for the isoleucine-valine (ilv) operons, we have isolated bacteriophages carrying the his and the ilv regions of the Escherichia coli chromosome. Transposition of the his operon to a site close to the att80 region of the E. coli chromosome has been carried out selecting for integration of a temperature-sensitive F'his(+) in the tonB locus. This transposed strain has been lysogenized with phi80i(lambda). Upon induction of the lysogen, His(+) transductants have been isolated, which, on further induction give rise to HFT (high frequency of transduction) lysates. Preliminary characterization of the transducing phage is reported. The ilv operon, carried on an F' particle, has been fused to an episome carrying the att80 region. The fused episome has been lysogenized with phi80i lambdat68. Upon induction of the lysogen, Ilv(+) transductants have been isolated which on further induction give rise to HFT lysates.     

Isolation of a λdv Plasmid Carrying the Bacterial gal Operon

A lambdadvgal plasmid carrying genes for controlled plasmid replication from phage lambda and the bacterial gal operon was isolated as a deletion mutant of phage lambdagalq4, which carries the gal operon between lambda genes P and Q. The plasmid DNA was found in cell extracts as covalently closed circular molecules. The plasmid was characterized by using genetic crosses, digestion with the specific endonuclease EcoRI, sucrose gradient centrifugation, and electron microscopy. In one clone analyzed, the plasmid was a complete dimer (O(lambda)P(lambda)galO(lambda)P(lambda)gal); in a subclone derived from it, the plasmid was a partial dimer with only one copy of gal (O(lambda)P(lambda)O(lambda)P(lambda)gal). The partial dimer may be a recombination product of the complete dimer, since test crosses show that the gal and lambda sequences in the plasmid can be separated by recombination. Analyses of the EcoRI digests of plasmid DNAs indicated one cleavage site per lambda gene sequence and none in the gal operon. A lambdadvgal monomer was approximately 6.7 x 10(6) daltons and the lambda gene and gal components were 3.9 x 10(6) and 2.8 x 10(6) daltons, respectively. The lambdadvgal plasmid can be introduced into a new bacterial host by transfection at an efficiency of 10(-6) per DNA molecule.     

Interaction of super-repressible and dominant constitutive mutations for the synthesis of galactose pathway enzymes in Saccharomyces cerevisiae.

Two dominant uninducible mutant alleles in the gal80 locus were identified. The GAL80s-1 and GAL80s-2 mutants showed novel phenotypes in response to the newly isolated GAL81-1 mutant allele, a dominant constitutive mutation linked to the gal4 locus; the GAL80s-1 GAL81-1 strain was inducible and the GAL80s-2 GAL81-1 strain was uninducible. Many galactose positive revertants from the GAL80s-2 GAL81-1 strain were isolated. It was proved that each revertant was due to a secondary mutation either in the gal80 or GAL81 locus, whereas revertants due to mutation at the supposed controlling site for the structural gene cluster of the galactose-pathway enzymes have not been isolated.     

Spontaneous Point Mutations That Occur More Often When Advantageous than When Neutral

Recent reports have called into question the widespread belief "that mutations arise continuously and without any consideration for their utility" (in the words of J. Cairns) and have suggested that some mutations (which Cairns called "directed" mutations) may occur as specific responses to environmental challenges, i.e., they may occur more often when advantageous than when neutral. In this paper it is shown that point mutations in the trp operon reverted to trp+ more frequently under conditions of prolonged tryptophan deprivation when the reversions were advantageous, than in the presence of tryptophan when the reversions were neutral. The overall mutation rate, as determined from the rates of mutation to valine resistance and to constitutive expression of the lac operon, did not increase during tryptophan starvation. The trp reversion rate did not increase when the cells were starved for cysteine for a similar period, indicating that the increased reversion rate was specific to conditions where the reversions were advantageous. Two artifactual explanations for the observations, delayed growth of some preexisting revertants and cryptic growth by some cells at the expense of dying cells within aged colonies, were tested and rejected as unlikely. The trp+ reversions that occurred while trp- colonies aged in the absence of tryptophan were shown to be time-dependent rather than replication-dependent, and it is suggested that they occur by mechanisms different from those that have been studied in growing cells. A heuristic model for the molecular basis of such mutations is proposed and evidence consistent with that model is discussed. It is suggested that the results in this and previous studies can be explained on the basis of underlying random mechanisms that act during prolonged periods of physiological stress, and that "directed" mutations are not necessarily the basis of those observations.     

Isolation of Plaque-Forming, Galactose-Transducing Strains of Phage Lambda

Plaque-forming, galactose-transducing lambda strains have been isolated from lysogens in which bacterial genes have been removed from between the galactose operon and the prophage by deletion mutation.—A second class has been isolated starting with a lysogenic strain which carries a deletion of the genes to the right of the galactose operon and part of the prophage. This strain was lysogenized with a second lambda phage to yield a lysogen from which galactose-transducing, plaque-forming phages were obtained. These plaque-forming phages were found to be genetically unstable, due to a duplication of part of the lambda chromosome. The genetic instability of these partial diploid strains is due to homologous genetic recombindation between the two identical copies of the phage DNA comprising the duplication. The galactose operon and the duplication of phage DNA carried by these strains is located between the phage lambda P and Q genes.     

Behavior of coliphage lambda in hybrids between Escherichia coli and Salmonella

Salmonella typhosa hybrids able to adsorb lambda were obtained by mating S. typhosa recipients with Escherichia coli K-12 donors. After adsorption of wild-type lambda to these S. typhosa hybrids, no plaques or infective centers could be detected. E. coli K-12 gal(+) genes carried by the defective phage lambdadg were transduced to S. typhosa hybrids with HFT lysates derived from E. coli heterogenotes. The lysogenic state which resulted in the S. typhosa hybrids after gal(+) transduction differed from that of E. coli. Ability to produce lambda, initially present, was permanently segregated by transductants of the S. typhosa hybrid. S. typhosa lysogens did not lyse upon treatment for phage induction with mitomycin C, ultraviolet light, or heat in the case of thermoinducible lambda. A further difference in the behavior of lambda in Salmonella hybrids was the absence of zygotic induction of the prophage when transferred from E. coli K-12 donors to S. typhosa. A new lambda mutant class, capable of forming plaques on S. typhosa hybrids refractory to wild-type lambda, was isolated at low frequency by plating lambda on S. typhosa hybrid WR4254. Such mutants have been designated as lambdasx, and a mutant allele of lambdasx was located between the P and Q genes of the lambda chromosome. Plaques were formed also on the S. typhosa hybrid host with a series of lambda(i21) hybrid phages which contain the N gene of phage 21. The significance of these results in terms of Salmonella species as hosts for lambda is discussed.     

Insertion mutations in the control region of the Gal operon of E. coli. I. Biological characterization of the mutations

Summary Galactose negative mutations are described which reduce the maximum expression of all three gal genes about 100-fold. The residual enzyme synthesis is not or only slightly inducible.These pleiotropic mutations map in the control region of the gal operon. No recombination is observed between these mutations. All mutants revert spontaneously to a Gal⁺ phenotype. In some mutations wildtype-like as well as constitutive revertants are obtained. The frequency of reversion can be increased by nitrosoguanidine (NG) in all mutants. The revertants, induced by this mutagen, are of a constitutive type.     

Mutations in the galactose-operator

Summary Constitutive mutations in the galactose operator in E. coli arise with a frequency ten times smaller than in the regulator gene. The operator constitutive mutations do not arise as a consequence of mutagen treatment.Operator constitutive mutations do not revert to wildtype spontaneously or after mutagen treatment.It is concluded that operator constitutive mutations are multisite mutations, and that the operator region is considerably smaller than the regulator gene in the gal operon.     

Interaction of super-repressible and dominant constitutive mutations for the synthesis of galactose pathway enzymes in Saccharomyces cerevisiae

Summary Two dominant uninducible mutant alleles in the gal80 locus were identified. The GAL80 ^s-1 and GAL80 ^s-2 mutants showed novel phenotypes in response to the newly isolated GAL81-1 mutant allele, a dominant constitutive mutation linked to the gal4 locus; the GAL80 ^s-1 GAL81-1 strain was inducible and the GAL80 ^s-2 GAL81-1 strain was uninducible. Many galactose positive revertants from the GAL80 ^s-2 GAL81-1 strain were isolated. It was proved that each revertant was due to a secondary mutation either in the gal80 or GAL81 locus, whereas revertants due to mutation at the supposed controlling site for the structural gene cluster of the galactose-pathway enzymes have not been isolated.This study was supported in part by grant no. 048164 to Y. Oshima from the Scientific Research Fund of the Ministry of Eduction, Japan     

Plasmid vectors for positive galactose-resistance selection of cloned DNA in Escherichia coli

Insertion of a HindIII-EcoRI fragment carrying part of the gal operon from lambda gal+ into pBR322 yields a plasmid (pAA3) which confers strong galactose sensitivity on E. coli strains deleted for the gal operon. Sensitivity to galactose is caused by the expression of kinase and transferase (but not epimerase) genes from a promoter located in the tet gene of pBR322. Insertion of a DNA fragment carrying Tn9 at the HindIII junction blocks gal expression and produces a galactose-resistant phenotype. Hence, galactose resistance can be used to select DNA fragments cloned at the HindIII site. The system was used efficiently for cloning lambda, yeast, and human DNA. The cloned fragments can be screened directly for the presence of promoters by testing for tetracycline resistance. Alternatively, these plasmids can be used as cosmids for cloning large fragments of DNA at a number of sites. Construction of several related vectors is described.     

Activation of mutated simian virus 40 enhancers by amplification of wild-type enhancer elements.

We show that duplication of any one of three separate simian virus 40 enhancer elements, A, B, or C, can compensate for loss of function in the remaining two. Simian virus 40 revertants containing point mutations within the A and C (dpm16) or B and C (dpm26) enhancer elements contain tandem duplications that include the remaining wild-type element. These simple tandem duplications can create enhancers 25-fold more active than that of the parental mutant. These revertants can arise by illegitimate recombination between heterologous viral genomes. This was demonstrated by the recombinants resulting from a mixed infection with the viruses dpm16 and dpm2, which contain mutations in the A and C elements and the B element, respectively.     

Efficient introduction of cloned mutant alleles into the Escherichia coli chromosome.

An efficient method for moving mutations in cloned Escherichia coli DNA from plasmid vectors to the bacterial chromosome was developed. Cells carrying plasmids that had been mutated by the insertion of a resistance gene were infected with lambda phage containing homologous cloned DNA, and resulting lysates were used for transduction. Chromosomal transductants (recombinants) were distinguished from plasmid transductants by their ampicillin-sensitive phenotype, or plasmid transductants were avoided by using a recBC sbcB E. coli strain as recipient. Chromosomal transductants were usually haploid when obtained in a nonlysogen because of selection against the lambda vector and partially diploid when obtained in a lysogen. Pure stocks of phage that carry the resistance marker and transduce it at high frequency were obtained from transductant bacteria. The lambda-based method for moving mutant alleles into the bacterial chromosome described here should be useful for diverse analyses of gene function and genome structure.