Isolation of Pure lac Operon DNA*

Nitrofurans, a class of antibacterial drugs in, extensive use, interferes with gene expression in a highly specific manner. While in the low dose range (0.5-25 ;&g/ml), 5-nitro-2-furfurylidene-l-aminohydantoin has no effect on transcription, it inhibits specifically the expression of one class of genes in translation. The specific inhibition concerns the inducible genes. The inhibition of messenger RNA expression occurs at the initiation step. The action of nitrofurans, thus, indicates heterogenei in e population of mRNA molecules and in the translational machinery, and suggests the possibility of selective translational control.

Section: Nf Action In Vitromentioning

confidence: 99%

Nitrofurans, a group of synthetic antibiotics, with a new mode of action: discrimination of specific messenger RNA classes.

Herrlich¹,

Schweiger²

1976

“…pRPG16 contains trpR on a 1.3-kb chromosomal BamHI fragment in pACYC177. Phage Aplac5 contains lacZ and lacY (15).…”

mentioning

confidence: 99%

Trp aporepressor production is controlled by autogenous regulation and inefficient translation.

Kelley¹,

Yanofsky²

1982

We constructed a trpR-lacZ gene fusion that specifies a hybrid protein that has full P-galactosidase activity. The gene fusion was associated with the unaltered trpR transcription and translation control region; thus, hybrid f-galactosidase production was an indicator of expression of the trp aporepressor (trpR) operon. To facilitate in vivo expression studies, a DNA segment containing the trpR-lacZ gene fusion and the trpR controlling region was transferred to bacteriophage A and subsequently inserted into the bacterial chromosome. Analyses of hybrid (-galactosidase production showed that the trpR operon is regulated autogenously but that the rate of synthesis of aporepressor varies only 4-to 5-fold in response to changes in the intracellular concentration of tryptophan. Under comparable conditions, the trp operon is regulated by trp repressor -70-fold. Therefore, the operators of the trp operon and the trpR operon must have very different affinities for trp repressor in vivo. The promoter controlling t-pR expression was found to be moderately active. Nevertheless, there are only about 50-300 molecules oftrp aporepressor per cell. The low aporepressor level appears to be due to inefficient translation of trpR mRNA.The trp repressor of Escherichia coli regulates at least three unlinked operons: trp, aroH, and trpR (1-4). The trp operon codes for the five polypeptides that catalyze the terminal sequence of reactions in tryptophan formation (5), aroH codes for one of three isozymes that perform the initial reaction of the common pathway of aromatic amino acid biosynthesis (6), and trpR codes for the trp aporepressor itself (7). The trpR operon thus is autogenously regulated, and its polypeptide product controls transcription initiation in two biosynthetic operons. The promoters of the three operons contain homologous operators, as expected (4).Previous indications that the trpR operon was autogenously regulated were based on in vitro analyses oftrp repressor binding to trpR operator DNA (4). In this report, we examine regulation ofthe trpR operon in vivo. Using a trpR-lacZ gene fusion that specifies a fully active hybrid f8-galactosidase, we show that the trpR operon is autoregulated 4-to 5-fold in response to changes in the intracellular tryptophan concentration. We also demonstrate that, although the trpR promoter is moderately active, inefficient translation of trpR mRNA results in low cellular levels of trp aporepressor. METHODS Growth Conditions. All cells were grown in minimal medium (8) supplemented with 0.2% glucose, 0.2% lactose, 0.3% glycerol, 0.1-0.5 mM isopropyl-,B3D-thiogalactoside, L-tryptophan at 50 k±g/ml, L-proline at 50 ,ug/ml, kanamycin at 30 Ag/ml, or ampicillin at 50 Ikg/ml as indicated. All temperature-sensitive lysogens were grown at 30-32TC.DNA Manipulations. Methods for cloning DNA fragments have been described (9). Restriction enzymes (New England BioLabs and Bethesda Research Laboratories) were used according to the supplier's instructions. Plasmid DNA was isolated from transfor...

“…Here also, an intervening nonhomologous region does (6). Region depicts E. coli DNA and region depicts the portion of the lac operon that is included on the Aplac5 phage used for these experiments (9).…”

Section: Discussionmentioning

confidence: 99%

“…The bacteria and bacteriophages used are listed in Table 1. Phages AlacZ36, AlacZ118, and AlacZ813 are derivatives of Aplac5 (5,9). Hybrid phages hyl and hy5 (6) were kindly provided by C. Radding.…”

mentioning

confidence: 99%

Indirect stimulation of genetic recombination

Golub

Low

1983

Recombination between lacZ alleles in crosses of AlacZ-X AlacZ-and F lacZ-X AlacZ- UV-irradiated Aphage which carries no lac region. This indirect stimulation is not dependent on induction of the SOS system. The bacterial uvr system can effectively remove the damages on the A DNA which cause the indirect stimulation. Among a number of mutations tested, only ssb-i was found to cause a drastic decrease in the indirect stimulation. Indirect stimulation was caused only by using phage that had a region of homology with the recombining phage. The homologous region can be separated from the recombining region by an extended'nonhomologous region (>7.9 x i03 base pairs). This implies that damages to the DNA molecule, which stimulate recombination, can be located very far from the recombining region of the molecule.In spite of considerable progress achieved lately in the study of genetic recombination in microorganisms, in general this process is still replete with mystery and puzzles. One of these puzzles is the well-known phenomenon of the stimulation of recombination by DNA damage. For example, it was shown that UV irradiation can increase considerably the frequency of recombination in many organisms (1). This phenomenon, which has been known for several decades, is still without a clear explanation. Two natural hypotheses for partial explanation of stimulation are: (i) the production of damage in the region of recombination, which can facilitate recombination by breakage and reunion, and (ii) induction of the bacterial SOS system that leads to excess synthesis of recA protein or other proteins (or both) which could be involved in recombination. However, our results show that, at least in our systems, stimulation of recombination by UV light can occur in the absence of either of these two conditions.We have studied recombination in the lacZ gene by crossing AplacZ-with either a different AplacZ-or a chromosomal lacZin bacteria lysogenic for phage A. In both systems, UV irradiation of a AplacZ-phage prior to infection leads to substantial increase in its subsequent recombination (2). To our surprise we find similar stimulation if UV-irradiated k+ infects bacteria simultaneously with unirradiated recombining (i.e., AlacZ-) phages. This indirect stimulation can be observed only when the damaged phage has a region of homology with the recombining phage. However, this region of homology may be removed from the recombining region by an extended region [7.9 kilobases (kb)] of nonhomology. We Recombination Systems. We used three systems of recombination between transducing phages, either with each other or with homologous regions on the bacterial chromosome. In all cases, a homoimmune resident prophage prevented replication and expression of phage functions. One system utilizes bacteria that are F-Alac(AcIind-) (i.e., lysogenic for AcIind-which is noninducible by UV or UV-damaged DNA) infected with two transducing phages AlacZ-which carry two different ochre mutations in lacZ. The efficiency of recombination is m...