Resistance to the peptide-like antibiotic negamycin in Escherichia coli

An $\text{Escherichia coli}$ mutant resistant to the peptide-like antibiotic negamycin carries a mutation, NEG40, which maps at minute 65 on the bacterial genome. Termination of protein synthesis, which is normally blocked by negamycin in wild type cellular extracts, continues with cellular extracts from the mutant in the presence of the drug; indeed, release of complete peptides from the polysomes still proceeds over a wide range of drug concentrations. The data suggest that the NEG40 mutation affects one of the components of the termination complex (ribosome or release factor).     

Ribosomal assembly deficiency in an Escherichia coli thermosensitive mutant having an altered L24 ribosomal protein.

Localized P1 mutagenesis was used to screen for conditionally lethal mutations in ribosomal protein genes. One such mutation, 2859mis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and cotransduces at 98% with rpsE (S5). The 2869mis mutation leads to thermosensitivity and impaired assembly in vivo of 50 S ribosomal particles at 42 °C. The strain carrying the mutation has an altered L24 ribosomal protein which at 42 °C shows weaker affinity for 23 S RNA than the wild-type protein. The mutational alteration involves a replacement of glycine by aspartic acid in protein L24 from the mutant. We conclude therefore that the 2859mis mutation affects the structural gene for protein L24 (rplX).     

Porcine D-amino acid oxidase: production of the biologically active enzyme in Escherichia coli

DNA molecules coding either for mature porcine D-amino acid oxidase or for truncated forms of the enzyme have been obtained by stepwise addition of synthetic oligonucleotides to a partial cDNA. Under the control of the lambda PL thermoregulatable promoter, these DNAs were respectively expressed in Escherichia coli as 36, 28 and 25 kilodalton polypeptides, specifically recognised by antibodies raised against the natural enzyme. None of the truncated proteins were biologically active whereas the mature recombinant species was able to hydrolyze D-alanine in vitro as efficiently as the natural product.     

Characterization of glycogen-synthase phosphatase and phosphorylase phosphatase in subcellular liver fractions

Upon fractionation of a postmitochondrial supernatant from rat liver, the synthase phosphatase (EC 3.1.3.42) activity (assayed at high tissue concentrations) was largely recovered in the glycogen fraction and to a minor extent in the cytosol. In contrast, the phosphorylase phosphatase (EC 3.1.3.17) activity was approximately equally distributed between these two fractions, a lesser amount being recovered in the microsomal fraction. The phosphatase activities in the microsomal and glycogen fractions were almost completely inhibited by a preincubation with the modulator protein, a specific inhibitor of type-1 (ATP,Mg-dependent) protein phosphatases. In the cytosolic fraction, however, type-2A (polycation-stimulated) phosphatase(s) contributed significantly to the dephosphorylation of phosphorylase and of in vitro phosphorylated muscular synthase. Liver synthase b, used as substrate for the measurement of synthase phosphatase throughout this work, was only activated by modulator-sensitive phosphatases. Trypsin treatment of the subcellular fractions resulted in a dramatically increased (up to 1000-fold) sensitivity to modulator, a several-fold increase in phosphorylase phosphatase activity and a complete loss of synthase phosphatase activity. Similar changes occurred during dilution of the glycogen-bound enzyme. A preincubation with the deinhibitor protein, which is known to counteract the effects of inhibitor-1 and modulator, increased several-fold the phosphorylase phosphatase activity, but exclusively in the cytosolic and microsomal fractions. It did not affect the synthase phosphatase activity. Taken together, the results indicate the existence of distinct, multi-subunit type-1 phosphatases in the cytosolic, microsomal and glycogen fractions.     

An assessment of the importance of intralysosomal and of alpha-amylolytic glycogenolysis in the liver of normal rats and of rats with a glycogen-storage disease

Mechanisms of glycogenolysis have been investigated in a comparative study with Wistar rats and gsd rats, which maintain a high glycogen concentration in the liver as a result of a genetic deficiency of phosphorylase kinase. In Wistar hepatocytes the rate of glycogenolysis, as modulated by glucagon and by glucose, was proportional to the concentration of phosphorylase a. In suspensions of gsd hepatocytes the rate of glycogenolysis was far too high as compared with the low level of phosphorylase a; in addition, only a minor fraction of the glycogen lost was recovered as glucose and lactate, owing to the accumulation of oligosaccharides. When the gsd hepatocytes were incubated in the presence of an inhibitor of alpha-amylase (BAY e 4609) glycogenolysis and the formation of oligosaccharides virtually ceased; the production of glucose plus lactate, already modest in the absence of BAY e 4609, was further decreased by 40%, owing to the suppression of a pathway for glucose production by the successive actions of alpha-amylase and alpha-glucosidase. Evidence was obtained that gsd hepatocytes are more fragile, and that amylolysis of glycogen occurred in damaged cells and/or in the extracellular medium. This may even occur in vivo, since quick-frozen liver samples from anesthetized gsd rats contained severalfold higher concentrations of oligosaccharides than did similar samples from Wistar rats. However, administration of a hepatotoxic agent (CCl4) caused hepatic glycogen depletion in Wistar rats, but not in gsd rats. The administration of phloridzin and of vinblastine, which have been proposed to induce glycogenolysis in the lysosomal system, did not decrease the hepatic glycogen level in gsd rats. Taken together, the data indicate that only the phosphorolytic degradation of glycogen is metabolically important, and that alpha-amylolysis is an indication of an increased fragility of gsd hepatocytes, which becomes prominent when these cells are incubated in vitro.     

The nature of the decreased activity of glycogen synthase phosphatase in the liver of the adrenalectomized starved rat

We have investigated the nature of the decrease in synthase phosphatase activity which occurs progressively in the livers of adrenalectomized rats that are starved for 48h. No evidence could be found for the accumulation of an inhibitor. Addition of the heat-stable deinhibitor protein, which antagonizes the effects of thermostable inhibitor proteins (inhibitor-1 and modulator), did not affect the activity of synthase phosphatase in gel-filtered liver extracts from normal or adrenalectomized starved rats; it did, however, increase the activity of phosphorylase phosphatase about fivefold in either condition. The restoration of synthase phosphatase activity by cortisol in vivo was prevented by actinomycin D. Further evidence concerning the nature of the missing protein came from a comparison of synthase phosphatase activities in liver homogenates from control and adrenalectomized starved rats, with the use of three distinct synthase b substrates. The apparent loss of synthase phosphatase activity in the deficient homogenates varied between 30% and 90% according to the type of substrate. The magnitude of this decrease corresponds to the degree of dependence of these substrates on the G-component of synthase phosphatase for efficient conversion to the alpha-form. No G-component could be isolated from livers of adrenalectomized starved rats. Cross-combination of subcellular fractions from control and deficient livers revealed an almost total loss of G-component, with little loss of S-component. This specific loss of functional G-component is identical to the deficiency previously observed in the livers of rats with severe chronic alloxan-diabetes.     

Amber mutations in Escherichia coli essential genes: isolation of mutants affected in the ribosomes.

Summary A method to obtain amber mutations in ribosomal protein genes is described. It relies on the P1-mediated localized mutagenesis (Hong and Ames, 1971) and on the fact that the recipient strain contains (a) an efficient but genetically unstable suppressor, (b) a particular thermoinducible prophage which kills suppressor hosts at 42° C. Exposure of these bacteria to the high temperature yields frequent suppressor-free derivatives while none will be found if the strain carries an amber mutation in an essential gene. Eleven mutants have been isolated by this method, of which at least six appear to carry amber mutations. All of them map close to, and to the right of spcA, in a region which codes mostly for ribosomal proteins. Three mutants were studied biochemically; all three show defective ribosomal assembly in vivo upon loss of suppression.