Mutations that alter the ebgA gene so that the evolved f-galactosidase (ebg) enzyme of Escherichia coli can hydrolyze lactose fall into two classes: class I mutants use only lactose, whereas class U mutants use lactulose as well as lactose. Neither class uses galactosylarabinose effectively. In this paper we show that when both a class I and a class II mutation are present in the same ebgA gene, ebg enzyme acquires a specificity for galactosylarabinose. Although galactosylarabinose utilization can evolve as the consequence of sequential spontaneous mutations, it can also evolve via intragenic recombination in crosses between class I and class II ebgA+ mutant strains. We show that the sites for class I and class II mutations lie about 1 kilobase, or about a third of the gene, apart in ebgA. Implications of these findings with respect to the evolution of new metabolic functions are discussed.The ebg (evolved f3-galactosidase) system of Escherichia coli is being employed as a model system to study the evolution of new metabolic functions. This model system has demonstrated, in the laboratory, the requirement for both structural and regulatory gene mutations for the evolution of a particular new metabolic function (1-3). It has also been used to demonstrate the existence of an evolutionary pathway, in that three sequentially selected spontaneous mutations in a structural gene were required for the evolution of a particular new metabolic function (4). We now turn our attention to the role of intragenic recombination in the evolution of new metabolic functions.Strains of E. coli that bear deletions within the lacZ (,B-galactosidase) gene, but in which the lacY (lactose permease) gene is intact, are unable to utilize lactose or other ,3-galactoside sugars as sole carbon and energy sources. A second /3-galactosidase, enzyme ebgO, is the product of the wild-type allele of the ebgA gene located at 66 min on the E. coli K-12 map (5, 6). Expression of the ebgA gene is under control of the tightly linked ebgR gene, which specifies a repressor (7). The wild-type enzyme, ebg°, has little activity toward natural fl-galactoside compounds, and even ebgR -strains, which synthesize about 5% of their soluble protein as ebg°enzyme, are unable to utilize lactose, lactulose, or galactosylarabinose (Gal-Ara) (2, 4). Spontaneous single-point mutations can evolve the wild-type allele, ebgA°, to ebgA +, resulting in enzyme with greatly increased activity toward lactose (8).Previous studies (2, 4) have shown that a number of different mutations in the ebgA gene can lead to enzyme with increased (3-galactosidase activity. Selection for lactose utilization results in two classes of ebgA + mutants: class I strains grow rapidly on lactose but are unable to utilize lactulose; class II strains grow more slowly on lactose and grow at a moderate rate on lactulose (4). Table 2 shows the first-order growth rate constants for representative members of each class. Studies of growth rates, coupled with kinetic analyses of purified enzymes, have...
The penicillin G acylase genes from the Proteus rettgeri wild type and from a hyperproducing mutant which is resistant to succinate repression were cloned in Escherichia coli K-12. Expression of both wild-type and mutant P. rettgeri acylase genes in E. coli K-12 was independent of orientation in the cloning vehicle and apparently resulted from recognition in E. coli of the P. rettgeri promoter sequences. The P. rettgeri acylase was secreted into the E. coli periplasmic space and was composed of subunits electrophoretically identical to those made in P. rettgeri. Expression of these genes in E. coli K-12 was not repressed by succinate as it is in P. rettgeri. Instead, expression of the enzymes was regulated by glucose catabolite repression.
Many of the challenges posed by the quantitative analysis of drug candidates in biological fluids are met by atmospheric pressure ionization tandem mass spectrometry (MS/MS). However, the development of suitable methodology requires the determination and optimization of many compound-specific instrumental variables. At a minimum, the m/z values of a precursor ion and a product ion, along with the optimum collision energy, must be known in order to construct a suitable method. MS/MS method performance is particularly sensitive to collision energy, which must be near-optimum for each compound analysed. Conventionally, these instrumental parameters are determined by continuous infusion of a standard solution into the ion source and optimizing each individual parameter. This paper describes the application of a technique based on the triple quadrupole mass spectrometer which streamlines the development of quantitative MS/MS methods. Received 10 January 1997; Revised 11 February 1997; Accepted 11 February 1997 Rapid. Commun. Mass Spectrom. 11, 593-597 (1997 Advances in molecular biology and combinatorial chemistry are revolutionizing drug discovery, yielding compounds with enhanced potency in ever-increasing numbers. The ability to measure rapidly compound levels in biological matrices has a huge impact on a given discovery program. Rapid analyte quantitation radically compresses the dispositional evaluation of compounds and adds significant momentum to the drug discovery process. Unfortunately, the development of suitable methods and subsequent sample analysis often requires a significant amount of time relative to other activities within a program. Matrices such as blood plasma, serum, tissue homogenates and urine contain literally thousands of small molecules and often contain high levels of protein and ionic material. The bioanalytical task is exacerbated by species and intersubject differences, variable fluidity and low compound levels. Traditionally, development of methods is rate limiting because extraction conditions and chromatography must be developed and optimized. Bioanalytical quantitation requires the application of significant capital and human resources for successful and rapid completion.The complexity of common biological matrices requires that analytical methodology be brought to bear which is (1) specific for the analyte of interest, (2) sufficiently sensitive for the particular assay methodology and (3) amenable to automation. The one analytical technique which most closely meets these criteria is high-performance liquid chromatography (HPLC) coupled with atmospheric pressure ionization mass spectrometry (HPLC/API-MS). This is widely used in the pharmaceutical industry for bioanalytical quantitation and is generally considered to be the technique of choice for the analysis of drugs and metabolites.1-5 Single-quadrupole instruments are in wide use for this application, which typically involves detection of a mass-selected molecular ion or other ion containing the relevant intact molecular species...
The ebg operon of Escherichia coli includes a second gene designated ebgB. The ebgB gene product is a 79,000-molecular-weight protein and is expressed coordinately with the ebgA gene product, ebg beta-galactosidase. Insertion of the transposable elements Tn5 and Tn9 into ebgA eliminates the expression of ebgB, suggesting that ebgB is distal to ebgA. Ultraviolet light mapping confirms that gene order. The function of the ebgB gene product is unknown.
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