Galactose Operon Research Articles

Expression of a bacterial gene turned on by a potent carcinogen

Contrary to mutagenesis, lysogenic induction produced by chemical carcinogens occurs in the majority of a population of lysogenic cells. Such a mass effect can therefore be measured at the biochemical level using an E. coli tester strain in which the galactose operon has been put under the negative control of the λ repressor. In this publication we show that galactokinase synthesis is turned on by aflatoxin B1 metabolites within an hour after treatment of the tester bacteria. Such a biochemical assay provides a useful means for identifying potential chemical carcinogens.

Dual control for transcription of the galactose operon by cyclic AMP and its receptor protein at two interspersed promoters

Our results demonstrate the existence of two initiation sites, S 1 and S 2, for transcription of the galactose operon of E. coli. Transcription from each of these sites responds to different regulatory mechanisms. In the presence of Cyclic AMP Receptor Protein (CRP) and cyclic AMP (cAMP), transcription initiates only at S 1. In the absence of CRP or cAMP, transcription starts from S 2. Examination of a gal promoter mutation shows that transcription from S 1 is abolished, but that initiation can occur at S 2 and is still subject to repression by CRP-cAMP. In vivo gal expression from this mutant promoter is actually increased when cyclic AMP or CRP are eliminated by mutations in the cya (adenylate cyclase) or crp genes. Studies of gal expression from a wild-type promoter in cya and crp mutants are also consistent with the use of the two startpoints in vivo and suggest that when CRP or cAMP is absent, transcription starts from S 2 in vivo. S 1 corresponds to the startpoint for CRP- and cAMP-dependent gal mRNA previously determined (Musso et al., 1977). S 2 precedes S 1 by 5 base pairs in the DNA of the gal regulatory region. A heptamer sequence analogous to those described for other promoters precedes each startsite by a distance of 6 base pairs. DNA sequence analyses of the gal promoter mutation mentioned above establish that the base change is in the second residue of the heptamer preceding S 1.

Determination of the nucleotide sequence of part of the regulatory region for the galactose operon from Escherichia coli.

We have determined the sequence of 59 base pairs in the DNA preceding the site for initiation of transcription in the galactose operon. DNA from a lambdagal transducing phage was digested with restriction endonucleases to obtain a DNA fragment from the gal regulatory region. This fragment extends from 59 base pairs prior to the transcription initiation site through the 45 base pairs which specify the 5'-terminal sequence of gal mRNA. Analyses of RNA transcripts derived from this fragment and a variety of direct DNA sequence analyses allowed us to deduce the following sequence for the DNA in this fragment: (formula: see text) Position +1 corresponds to the site for initiation of gal mRNA synthesized in the presence of cyclic AMP and its receptor protein, CRP. Transcription experiments indicate, however, that this fragment lacks some element of the gal promoter required for the stimulation of transcription by CRP-cAMP. There are, nonetheless, some similarities between the gal sequence preceding the transcription start site and the sequences of other promoter regions. These include the heptamer sequence T-A-T-G-G-T-T (--12 to --8) and the sequence A-C-A-C-T-T-T (--36 to --30).

Genetic inversion in the formation of an Hfr strain from a temperature-sensitive F' gal strain

An Hfr strain (PB15) that carries a duplicated copy of the galactose operon genes flanking the integrated sex factor is unusually stable since it does not show excision of the repeated deoxyribonucleic acid segment. The right-hand galactose operon is in the normal orientation. Deletion mutations that eliminate the right-hand galactose genes, the sex factor, and some of the left-hand operon have been isolated. Mutants believed to have their left-hand galactose operon inverted were able to be induced for galactose epimerase synthesis by D-fucose but did not show escape synthesis on induction of bacteriophage lambda. Ribonucleic acid specific for the galactose operon was isolated after induction of lysogenic strains presumed to carry the galactose operon in the normal and inverted orientation. Hybridization to the isolated left and right strands of lambdapgal showed that the noninformational strand of the left-hand galactose operon of the deletion mutant of PB15 was transcribed on escape induction. These results show that inversion has occurred.

Specialized transduction of the biotin region of Escherichia coli by phage T1

Phage T1 transduces Bio+ by special mechanism which leads to a higher efficiency of Bio+ transduction than other bacterial markers. Efficient Bio+ transduction depends on a site located between the galactose operon and the bacterial attachment site for phage λ. Evidence is presented which supports the hypothesis that the site is essential for efficient Bio+ transduction because at the site phage T1 initiates head filling in a polar (unidirectional) fashion leading to increased pickup of the Bio+ marker.

Isolation and characterization of Escherichia coli K-12 F- mutants defective in conjugation with an I-type donor

Escherichia coli K-12 F- mutants defective in conjugation with an I-type donor (ConI-) were isolated and characterized. These mutants are specific in that they are conjugation proficient with other types of donor strains. They have an altered susceptibility to phages and detergents. Chemical analysis of the cell envelopes of mutant strains has shown that the lipopolysaccharide (LPS) is altered and that one major outer-membrane protein is absent. Conjugation experiments in which LPS from wild-type cells was added to a mating mixture, made up with wild-type donor and recipient cells, showed inhibition in transconjugant formation when an I-type donor, but not an F-type donor, was used. This strongly suggests that LPS of the recipient cell is directly involved in the ability to mate with an I-type donor but not with an F-type donor. The mutations are located in the 78- to 82-min region of the E. coli map, with one exception where the mutation maps near or in the galactose operon.

Nucleotide sequence of the operator-promoter region of the galactose operon of Escherichia coli.

We have derived the nucleotide sequence of a segment of the operator-promoter region of the galactose operon of E. coli, by using a variety of DNA sequencing analyses. We have previously reported the sequence of the 5' terminal portion of gal mRNA [Musso, R. E., de Crombrugghe, B., Pastan, I., Sklar, J., Yot, P. & Weissman, S. (1974) Proc. Natl. Acad. Sci. USA 71, 4940-4944] and of the 59 base pairs preceding the startpoint of gal transcription (J. Sklar, S. Weissman, R. Musso, R. Di Lauro, & B. de Crombrugghe, submitted). In conjunction with those results, the present data provide the sequence of the gal operator-promoter region. This sequence is compared with similar sequences in other promoters and operators. Tentative mechanisms for the regulation of the galactose operon are discussed.

Biochemical construction of specific chimeric plasmids from ColE1 DNA and unfractionated Escherichia coli DNA.

A series of chimeric plasmids was constructed using colicinigenic factor E1 (ColE1) DNA as the replicon and DNA fragments carrying the galactose or tryptophan operons from E. coli. Restriction endonuclease EcoRI digests of ColE1 DNA and various DNAs containing the trp or gal operons were joined by T4 polynucleotide ligase [polynucleotide synthetase (ATP), poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase (AMP-forming), EC 6.5.1.1]. Chimeric plasmids carrying the desired genes were selected after transformation of Trp- or Gal- cells with ligated DNA. By using this method, we constructed ColE1-gal and ColE1-trp chimeric plasmids in which the source of the bacterial gal and trp operons was an unfractionated EcoRI digest of total E. coli DNA. The frequency of recovery of such chimeric plasmids is 10 to 20 colonies per mug of ligated DNA used in the transformation step. The method utilized in this report for constructing specific chimeric plasmids from total E. coli DNA is very simple. It requires only endonuclease R-EcoRI and T4 polynucleotide ligase, both of which are commercially available. The yield of transformants suggests that this method will be useful for cloning and amplifying a wide variety of functionally defined genes from E. coli and other prokaryotic organisms.

Microassay for UDP-galactose 4-epimerase activity

A sensitive microassay technique for determining UDP-galactose 4-epimerase activity in cell-free lysates has been devised. This method measures directly the conversion of UDP-galactose to UDP-glucose by thin-layer chromatographic separation of the two compounds. Radioactive labeling of the substrate serves as the quantitative basis for the assay. This method has been found to be approximately 100 times more sensitive than previously described spectrophotometric assay methods. Using the chromatographically based assay method a polar epimerase mutant and a promoter mutant of the galactose operon, which could not be distinguished enzymatically by the spectrophotometric assay method, could now be distinguished on the basis of their epimerase activity levels.

Reversion of the gal3 mutation of Escherichia coli: partial deletion of the insertion sequence.

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 (lambda) strain. Although the constitutive reversions could be transduced by lambda, 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 lambda. In order to facilitate the packaging of these reversions, the chlD-pgl region was deleted from the parent gal3 (lambda) strain. Unexpectedly, the gal3 mutation in the majority of these deletions reverted to produce stable, constitutive reversions exclusively. The explanation proposed was that the chlL-pgl deletions had also removed part of the gal operator-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 (lambda) strain, and not in gal+, gal+(lambda), or gal3 strains. Moreover, the frequency of spontaneous chlD-pgl deletions increased 10- to 15-fold in presence of the gal3 insertions. A lambdagal phage bearing a true stable, constitutive reversion (galc200) was isolated from the revertant strain by subsequent deletion of the chlD-pgl segment (delta31). Electron micrographs of lambdagal+ and lambdac200 delta31(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 lambda; and (iii) the gal3 insertion appears to inhibit the production of lambdagal 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.

Mechanism of reversion of the gal3 mutation of Escherichia coli

The gal3 mutation is an insertion of a DNA sequence in the operator-promoter region of the galactose operon of E. coli. It reverts spontaneously to produce three kinds of gal+ revertants, which are: (i) stable and inducible, (ii) stable and constitutive, and (iii) unstable and constitutive. The constitutive revertants also show drastically reduced frequencies of transduction with lambda. The mechanism by which these reversions occur has remained unknown. It is proposed that the stable and inducible revertants arise by accurate excision of the insertion sequence. The unstable and constitutive revertants arise by tandem duplications of the gal operon in such a way that the structural genes of the extra copy of gal operon become connected to a different promoter. The resulting tandem configuration (gal3 ETK...P'E'T'K') permits constitutive expression and gal3 segregation (by internal recombination) simultaneously. The proposal was tested by comparison of the buoyant densities in CsCl of derivatives of a lambdagal phage carrying gal+, gal3, and the inducible and constitutive revertants. The densities of the inducible revertants were identical to the wild type, and the slight increase in density found to be associated with the gal3 insertion was missing. It was concluded that inducible revertants arise by excision of the inserted sequence. In contrast, lysates of a constitutive revertant exhibited several anomalous properties. The lysates contained a small quantity of phage whose density was identical to lambdagal3, produced few gal+ transductants (10(-3)-10(-4) of a normal HFT lysate), and the transductants were stable and constitutive. In turn, these abnormal transductants produced lysates which showed no lambdagal particles on centrifugation, and no transducing activity whatsoever. These anomalous properties of the constitutive revertant were attributed to the failure of lambda to package the DNA duplication efficiently. Transduction experiments with P1 (which can package more DNA than lambda) show that the unstable, constitutive reversions were located adjacent to prophage lambda. Segregation of the gal and lambda markers among the gal+ transductants was in accordance with the pattern expected for a duplication. Introduction of a recA marker resulted in stabilization of the reversion without affecting its constitutive expression. It was concluded that the unstable, constitutive reversion was a tandem duplication. It is further proposed that the stable, constitutive class of revertants might represent inverted (gal3 ETK...K'T'E'P') or partial tandem (gal3 ET...E'T'K') duplications of the gal operon.

The nature of the gal3 mutation of Escherichia coli

The gal3 mutation of the galactose operon of E. coli, first described by Morse, Lederberg, and Lederberg in 1956, exhibits several unique and unusual properties. This mutation originated spontaneously and also reverts spontaneously, but fails to respond to various chemical mutagens. It is extremely polar, but the polarity is not relieved by nonsense suppressors. The gal+ revertants are of at least two different kinds. One class of revertants is stable and exhibits inducible enzyme synthesis, whereas the other class is unstable and constitutive. The properties of gal3 can be explained by the assumption that this mutation was caused by the insertion of a DNA sequence in the operator-promoter region of the gal operon. This proposal was tested by the construction of a gal3 derivative of a lambdagal+ phage and comparison of the physical properties of the two phages. Density-gradient centrifugation studies indicated that the gal3 mutation was accompanied by a 0.0033 g/ml increase in the buoyant density of the phage. This increase in density corresponds to the addition of 1200 base pairs. This observation was confirmed by electron microscopy of lambdagal+/lambdagal3 heteroduplexes. A single-stranded addition loop, 1160 base pairs in length, was found at a fixed position. It was concluded that the gal3 mutation was caused by the insertion of a sequence similar to IS3 in length. These studies also permit an estimation of the physical size of the gal operon. It is found to be 3600 base pairs long, separated from the attlambda (B.P') site by approximately 14000 base pairs.

Regulation of galactose operon at the gal operator-promoter region in Escherichia coli K-12

The capR (lon) product controls expression of the gal operon independently of the galR repressor. Previously, mutations of the gal operon have been isolated that are semiconstitutive and alter response to the capR and/or capT product. Such mutants imply the existence of a distinct site in the operon that responds to capR (capT) control. This mutation could be either in a site near the operator-distal end of the galE gene, which signals rho factor termination of transcription in vitro or in a site in the operator-promoter region. Bacteriophage U3 was used to isolate galE mutations in HC2142 (a mutant exhibiting reduced response to capR control). P1 transduction was used to cross these mutants with a set of galE gene deletion. Analysis of the resulting Gal+ recombinants indicates that the regulatory site is in the operator-promoter region. Hence, it is unlikely that capR functions in control as an anti-rho factor at the operator-distal end of the galE gene, but more likely as previously suggested, at a second operator distinct from one responding to galR repressor control. Upon induction with D-fucose, a promoter mutant (UV211) isolated previously expressed 20 to 30% of the galactose enzymes that the wild type exhibited in the presence of the inducer D-fucose. The effects of various mutations in cya, capR, and galR on galactokinase synthesis in this mutant were determined. Galactokinase was derepressed by capR as well as galR, but the presence or absence of the cya gene product was unimportant.

Metabolism of messenger RNA from the gal operon of Escherichia coli

The size and metabolism of messenger RNA from the galactose ( gal) operon of Escherichia coli has been studied. The three known structural genes of the operon are about 1100 nucleotide pairs each and code for an epimerase ( E), transferase ( T) and kinase ( K) gene, in that order. (1) The full-length gal mRNA in both B and K12 strains is about 4500 nucleotides or 1200 longer than that predicted for these three genes. The transcription time to the end of the epimerase gene was estimated by the lag before appearance of transferase mRNA. These measurements are consistent with about 520 nucleotides of gal mRNA proximal to the transferase message. The position of the remaining additional nucleotides is not known. (2) The functional inactivation rate of each message was measured by following the decreasing rate of enzyme synthesis after a one-minute induction by fucose. The half-life for each message was: epimerase 1.0 minute, transferase 0.6 minute, and kinase 1.5 minutes. (3) Mass decay of gal mRNA, induced for one minute, was followed by hybridization to DNA from transducing λ gal phages. Messenger RNA from the operator-proximal half, operator-distal two-thirds or from the entire operon decays with a half-life of about 1.1 minutes. These data cannot determine whether or not mass decay of each message is proportional to its inactivation rate. (4) The size distributions of decaying gal mRNA were determined in sucrose gradients or polyacrylamide gels with time of decay. They show clearly that gal mRNA is attacked by endonucleolytic cleavage. Fragments of about 2200 nucleotide length accumulate and have the specificity of transferase-kinase mRNA. The rapid inactivation of transferase message may result from cleavage of the epimerase-transferase mRNA junction to generate these transferase-kinase pieces.

The 5'-terminal nucleotide sequence of galactose messenger ribonucleic acid of Escherichia coli.

The 5'-terminal sequence of mRNA from the galactose operon of E. coli has been determined. gal RNA is synthesized in vitro by means of a kinetically controlled purified transcription system and is isolated by a two-step RNA.DNA hybridization scheme. The following sequence of the first 77 nucleotides has been deduced by analysis of oligonucleotides produced by digestion with T(1), pancreatic, and carboxymethylated pancreatic ribonucleases: ppA-U-A-C-C-A-U-A-A-G-C-C-U-A-A-U-G-G-A-G-C-G-A-A-U-U-A-U-G-A-G-A-G-U-U-C-U-G-G-U-U-A-C-C-G-G-U-G-G-U-A-G-C-G-G-U-U-A-C-A-U-U-G-G-A-A-G-U-C-A-U-A-C-C-U-G-U. Residues 27-77 correspond to the amino terminal 17 amino acids of UDPgalactose 4-epimerase (EC 5.1.3.2), the protein specified by the promoter-proximal structural gene of the operon. A self-complementary sequence occurs near the 5' terminus; 12 of 15 nucleotides between residues 4 and 18 are symmetrically located about position 11. The sequence of residues 22-33 closely resembles part of the lactose operator sequence reported previously [Gilbert, W. & Maxam, A. (1973) Proc. Nat. Acad. Sci. USA 70, 3581-3584].

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

mRNA 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.

Role of cyclic adenosine 3',5'-monophosphate in the in vivo expression of the galactose operon of Escherichia coli.

Studies of levels of galactokinase in Escherichia coli with mutations affecting synthesis of, or response to, cyclic adenosine 3',5'-monophosphate show that this nucleotide does not play a major role in expression of the galactose operon, causing at most a twofold stimulation. The discrepancy between our in vivo results and the marked stimulation by cyclic adenosine 3',5'-monophosphate in in vitro systems indicates that current cell-free systems lack a factor which allows efficient expression of the galactose operon even in the absence of cyclic adenosine 3',5'-monophosphate or of the binding protein for this nucleotide.

Transcriptional control of the calactose operon by the capR (lon) and capT genes.

Mutations in capR or capT cause derepression of the enzymes of the gal operon. The gal-specific messenger ribonucleic acid is directly proportional to the gal enzyme levels in wild type, capR, and capT strains. These results indicate that capR and capT control the gal operon at the transcriptional level.

Biological and Molecular Evidence for the Transgenosis of Genes from Bacteria to Plant Cells

Specialized transducing phages (lambda and varphi80) have been used as vectors in the transfer of genes (wild type and mutant) from the bacterium Escherichia coli to haploid cell lines of the plants Lycopersicon esculentum and Arabidopsis thaliana. The overall phenomenon of transfer, gene maintenance, transcription, translation, and function has been termed transgenosis. Transgenosis of galactose and lactose operon genes was detected by survival and growth of the plant cells on defined medium with galactose and lactose as sole sources of bulk carbon. Phages carrying a defective operon, unrelated bacterial genes, or no bacterial genes, do not affect the normal result of death on these media, nor do they prevent growth on optimal growth media. Transgenosis of the E. coli gene z (lac operon) was confirmed by a biochemical-immunological test specific for E. coli beta-galactosidase. Plant cells were unable to effectively suppress an E. coli nonsense mutation. The E. coli mutant suppressor gene, supF(+), specifies insertion of tyrosine at amber (UAG) nonsense codons. Introduction of supF(+) results in a lethal transgenosis on medium normally optimal for plant cell growth. It is concluded that amber codons are vital to the life of plant cells. Differentiating cells of A. thaliana were not affected by supF(+).

Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli.

We have developed methods for covalently joining duplex DNA molecules to one another and have used these techniques to construct circular dimers of SV40 DNA and to insert a DNA segment containing lambda phage genes and the galactose operon of E. coli into SV40 DNA. The method involves: (a) converting circular SV40 DNA to a linear form, (b) adding single-stranded homodeoxypolymeric extensions of defined composition and length to the 3' ends of one of the DNA strands with the enzyme terminal deoxynucleotidyl transferase (c) adding complementary homodeoxypolymeric extensions to the other DNA strand, (d) annealing the two DNA molecules to form a circular duplex structure, and (e) filling the gaps and sealing nicks in this structure with E. coli DNA polymerase and DNA ligase to form a covalently closed-circular DNA molecule.