H. Saedler

Oo and strong-polar mutations in the gal operon and insertions

SummaryThree λ dg phages carrying strong-polar mutations in the gal operon are denser than the corresponding phages carrying the wildtype gal operon or reversions of the mutations to the Gal+ phenotype. The latter phages have the same density. It is concluded that these strong-polar mutations are insertions of DNA into the gal operon.The amount of inserted DNA is different in the three mutations and is calculated to be 450, 1,080 and 1,800 nucleotide pairs respectively.The strong-polar phenotype is also found in a mutant supplied by A. Taylor which carries a Mu-1 phage integrated into the transferase gene.

IS1 is involved in deletion formation in the gal region of E. coli K12

SummaryThe DNA sequence IS1, which is 800 pairs long, has been shown to integrate into various bacterial and phage operons. The presence of this DNA sequence in the gal operon of E. coli K12 leads to an 30–2000 fold increase in deletion formation in the gal region as compared to wildtype. This high frequency of deletion formation is specific for IS1 and is independent of the cellular recA function. While the frequency of reversion of gal::IS1 mutations, which also is independent of recA, is not affected by the growth temperature of the cells, the formation of deletions in the gal::IS1 system is strongly dependent on the temperature of growth. Mapping experiments showed that one endpoint of the deletions in most cases is at the site of the IS1 mutation and the second endpoint seems to be at various but preferred sites. The formation of the different classes of deletions observed is affected differently by the growth temperature of the cells. A model to account for these results is presented.

Multiple copies of the insertion-DNA sequences IS1 and IS2 in the chromosome of E. coli K-12

SummaryAbout 8 copies of the DNA sequence IS1 (which consists of 800 nucleotide pairs) and about 5 copies of the DNA sequence IS2 (1400 nucleotide pairs) have been detected by DNA-DNA hybridization in the chromosome of E. coli K-12. No homology is observed between IS1 and IS2. Both IS1 and IS2 are also found in the DNA of the F plasmid.

Strong-polar mutations in the transferase gene of the galactose operon in E. coli

SummaryA class of mutations in the transferase gene of the galactose operon in E. coli is described, which is strongly polar for the synthesis of kinase. The latter enzyme is made only to the extent of about 0.1% of the amount made in the induced wildtype. This amount is not dependent on the map position of the mutations and the residual synthesis is non-inducible. The mutants thus resemble 0° mutants in the same operon.Epimerase, which is coded for by the gene proximal to the transferase gene with respect to the operator, is made in normal amounts and its synthesis is normally inducible.The mutants do not seem to belong either to the nonsense or to the frameshift class on the basis of reversion pattern, suppressibility, and degree of polarity. The possible nature of the mutations is discussed.

Two insertions in the galactose operon having different sizes but homologous DNA sequences

SummaryDNA from λdg phages carrying the strong-polar insertions N 102 and N 116 was transcribed into RNA in vitro. The RNA was exhaustively hybridized to λdg-DNA carrying the wildtype galactose operon. The remaining RNA contained a fraction which specifically hybridized to DNA carrying the insertion.The insertion-specific RNA hybridizes equally well to both insertions, regardless from which insetion the RNA was transcribed. The amount of insertion-specific RNA transcribed from the larger insertion N 102 is 2–3 times larger than the amount transcribed from insertion N 116.

Oo mutations in the galactose operon in E. coli

SummaryEleven mutants lacking the three enzymes of galactose fermentation were investigated.Eight of the mutants revert spontaneously to the Gal+ phenotype. These cannot be deletions. Six of these spontaneously reverting mutants do not respond to the mutagens 2-aminopurine, ethyl-methanesulfonate and N-Methyl-N′-Nitro-N-nitrosoguanidine. It is concluded that these oo mutations cannot be reverted by base substitution.The eleven oo mutants are not of the amber or ochre type as shown by their behaviour towards suppressor genes.The possible nature of the mutations is discussed.Oo mutants in the galactose operon in E. coli were studied with respect to residual enzyme production. Six out of ten mutants did not produce detectable epimerase. All mutants produced transferase and kinase in amounts about 10-3 of the induced wildtype level. The residual enzyme synthesis is not inducible. It is concluded that the mutants have a dual effect: They reduce the maximum amount of enzyme synthesis by a factor of 103, and they are constitutive. The meaning of this finding is discussed.

Insertion mutations in the control region of the galactose operon of E. coli

SummaryGalactose 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.

Negative control of the galactose operon in E. coli

SummaryNon-inducible mutants have been isolated which synthesize the three galactose enzymes with the basal rate both in the absence and in the presence of inducers. These mutations are closely linked to the lysA gene, as are the constitutive mutations in the regulator gene first described by Buttin (1963).The non-inducible mutants are Gal− on EMB gal plates. Revertants to the Gal+ phenotpye are constitutive. Heterozygotes have been prepared at the locus of the regulator gene (galR), abd dominance studies involving the different alleles at this locus have been carried out. The non-inducible mutations are dominant over the wildtype, and this in turn is dominant over constitutive mutations in the galR gene.Starting from the non-inducible mutations, deletions have been isolated, which extend from the galR gene into the lysA gene. These are constitutive.The behavior of the non-inducible mutations and of the deletions are strong arguments for negative control of the galactose operon.

Polarity of amber mutations and suppressed amber mutations in the galactose operon of E. coli

SummaryAmber mutants in the t gene of the galactose operon have been examined for polarity in the presence and absence of the suppressors suI and suyMel.In the absence of suppressors there is a gradient of polarity with the more polar mutations nearer the epimerase gene. This polarity is cis-dominant. Amber t mutants have raised epimerase levels but this effect is recessive. The operon is normally inducible in the presence of amber mutations. A double amber mutant had the polarity of the mutation nearest the epimerase end of the gene.In the presence of suppressors there is practically no gradient of polarity. This is in disagreement with the model proposed by Martin et al. (1966) and Yanofsky and Ito (1966). Modifications of this model to fit the present data are proposed.

mRNA distal to polar nonsense and insertion mutations in the gal operon of E. coli

SummarymRNA of the galactose operon of E. coli was measured in wildtype E. coli and in gal operon amber and insertion mutants. The mRNA coded by the distal half of the operon is reduced in the mutants. This reduction is more pronounced in the insertion mutants than in the amber mutants. It was compared with the polar effects of the mutations on the enzymes of the operon.