Expression of Escherichia coli ribosomal protein and RNA polymerase genes cloned on plasmids.

Summary Fragments of drif ^d 18 DNA with different end-points within the set of structural genes of ribosomal proteins L11 (rplK), L1 (rplA), L10 (rplJ) and L12 (rplL) as well as the (rpoB) and (rpoC) subunits of RNA polymerase have been cloned on plasmids. These plasmids were transformed in host cells which were mutant for each of the genes, enabling expression of both wild-type (plasmid-borne) and mutant (chromosomal) genes to be differentiated. On the basis of these results we propose the following genetic structure for the region: rplK and rplA are in one operon; rplL, rpoB and rpoC are in a second. Our data suggest the possibility that rplJ is by itself in an operon situated between the other two.     

Polarity of amber mutations in ribosomal protein genes of Escherichia coli.

Two amber mutations have been mapped inside the spcA-strA region (now called rpsE-rpsL) on the bacterial genome. Derivatives of the transducing phage lambda fus3 carrying each mutation were constructed and assayed in ultraviolet-irradiated bacteria to identify the mutated genes and measure the polarity of the mutations. The data indicated that both mutations, 3162(Am) and 3161(Am), affect genes coding for ribosomal proteins: rplC (L3) and rpsN (S14), respectively. It was shown also that each mutation exerts, inside of its respective operon (S10 and spc units), a relatively strong polar effect on genes distal to the mutated locus.     

Expression of ribosomal protein genes cloned in a hybrid plasmid in Escherichia coli: gene dosage effects on synthesis of ribosomal proteins and ribosomal protein messenger ribonucleic acid.

Using ColE1-TnA hybrid plasmid RSF2124 as the cloning vector, we constructed a hybrid plasmid, pNO1001, which carried seven ribosomal protein (r-protein) genes in the spc operon together with their promoter. The plasmid also carried three r-protein genes which precede the spc operon, but did not carry the bacterial promoter for these genes. Expression of r-protein genes carried by pNO1001 was studied by measuring messenger ribonucleic acid and r-protein synthesis in cells carrying the plasmid. It was found that the messenger ribonucleic acid for all the promoter-distal r-protein genes was synthesized in large excess relative to messenger ribonucleic acid from other chromosomal r-protein genes which are not carried by the plasmid. However, only the two promoter-proximal r-proteins, L14 and L24, were markedly overproduced. The absence of large gene dosage effects on the synthesis of other distal proteins appeared to be due, at least in part, to preferential inactivation and/or degradation of the distal message which codes for these proteins; in addition, some preferential inhibition of translation of the distal message might also have been involved. Overproduced L14 and L24 were found to be degraded in recA+ strains at both 30 and 42 degrees C; in recA strains, the degradation took place at 42 degrees C but was very slow or absent at 30 degrees C. The recA strains carrying pNO1001 failed to form colonies at 30 degrees C, presumably because of overaccumulation of r-proteins. The results suggest that degradation of excess r-proteins is an important physiological process.     

Transcription of ribosomal protein genes carried on F'' plasmids of Escherichia coli.

Summary Spontaneous mutants of the petite-negative yeast Kluyveromyces lactis, resistant to the antibiotics chloramphenicol and oligomycin, were isolated and genetically characterized.Three chloramphenicol-resistant mutants showed non-Mendelian inheritance when crossed to sensitive parents.Of 5 oligomycin-resistant strains studied, three exhibited resistance due to the presence of an extrachromosomal mutation. The resistance of the other two deriving from a nuclear and recessive mutation.When two factor crosses in trans configuration were performed between two of the chloramphenicol and the five oligomycin-resistant mutants a polarity in recombination was observed with a predominance of sensitive (O^SC^S) over resistant (O^RC^R) reciprocal recombinants.Allelism tests carried out among the oligomycin-resistant mutants indicated the presence of at least two distinct extrachromosomal regions responsible for the resistance.     

Epitopes of Escherichia coli ribosomal protein S13

To analyze the immunochemical structure ofEscherichia coli ribosomal protein S13 and its organizationin situ, we have generated and characterized 22 S13-specific monoclonal antibodies. We used a competitive enzyme-linked immunosorbent assay to divide them into groups based on their ability to inhibit binding of one another. The discovery of five groups with distinct binding properties suggested that a minimum of five distinct determinants on S13 are recognized by our monoclonal antibodies. The locations of the epitopes detected by these monoclonal antibodies have been mapped on S13 peptides. Three monoclonal antibodies bind a S13 C-terminal 34-residue segment. All the other 19 monoclonal antibodies bind a S13N-terminal segment of about 80 residues. The binding sites of these 19 monoclonal antibodies have been further mapped to subfragments of peptides. Two monoclonal antibodies recognized S13_1–22; three monoclonal antibodies bound to S13_1–40; the binding sites of three other antibodies have been located in S13_23–80, with epitopes possibly associated with residues 40–80. The remaining 11 monoclonal antibodies did not bind to these subfragments. These data provide molecular basis to the structure of S13 epitopes, whosein situ accessibility may reveal the S13 organization on the ribosome.     

Isoaspartate in ribosomal protein S11 of Escherichia coli

Isoaspartyl sites, in which an aspartic acid residue is linked to its C-flanking neighbor via its beta-carboxyl side chain, are generally assumed to be an abnormal modification arising as proteins age. The enzyme protein L-isoaspartate methyltransferase (PIMT), present in many bacteria, plants, and animals, catalyzes the conversion of isoaspartate to normal alpha-linked aspartyl bonds and is thought to serve an important repair function in cells. Having introduced a plasmid into Escherichia coli that allows high-level expression of rat PIMT, we explored the possibility that the rat enzyme reduces isoaspartate levels in E. coli proteins, a result predicted by the repair hypothesis. The present study demonstrates that this is indeed the case; E. coli cells expressing rat PIMT had significantly lower isoaspartate levels than control cells, especially in stationary phase. Moreover, the distribution of isoaspartate-containing proteins in E. coli differed dramatically between logarithmic- and stationary-phase cultures. In stationary-phase cells, a number of proteins in the molecular mass range of 66 to 14 kDa contained isoaspartate, whereas in logarithmic-phase cells, nearly all of the detectable isoaspartate resided in a single 14-kDa protein which we identified as ribosomal protein S11. The near stoichiometric levels of isoaspartate in S11, estimated at 0.5 mol of isoaspartate per mol of S11, suggests that this unusual modification may be important for S11 function.     

Expression of Anabaena ferredoxin genes in Escherichia coli

The genes for ferredoxin from heterocysts (fdx H) and vegetative cells (pet F) of Anabaena sp. strain 7120 were subcloned into plasmid pUC 18/19. Both genes were expressed in Escherichia coli at high levels (10% of total protein). Pet F could be expressed from its own promoter. The ferredoxins were correctly assembled to the holoprotein. Heterocyst ferredoxin was purified from E. coli extracts on a large scale. Its biochemical and biophysical properties were identical to those of the authentic ferredoxin, isolated from Anabaena heterocysts.This paper is dedicated to Prof. A. Trebst on the occasion of his 60th birthday.     

Insertion of DNA carrying ribosomal protein genes of Escherichia coli into Charon vector phages.

DNA fragments from lambdaspc1 and lambdafus2, carrying ribosomal protein genes from Escherichia coli, were inserted into lambda phage vectors Charon 3 and Charon 4. Eight of the resulting clones were characterized by agarose gel electrophoresis of EcoRI digests, analytical CsCl equilibrium centrifugation, and electron micrographic analysis of heteroduplexes. In each case, the identity, order, and orientation of each cloned fragment was determined. In all, 8 of the 12 EcoRI fragments of lambdafus2 were cloned in various arrangements. In the accompanying paper, genes for 15 ribosomal and related proteins and three bacterial promoters were detected in these phages. In addition, four of the hybrid phages carried fragments of lambda-DNA including the phage origin of replication (ori), the late promoter, PR', and the cohesive ends (cos site) in both orientations. The latter phages yield a circularly permuted collection of DNA molecules.     

Isolation and characterization of specialized transducing lambda phages carrying ribosomal protein genes of Escherichia coli

Summary Insertion in an episome of a kanamycine-resistant element (Tn5) at the polynucleotide phosphorylase gene level, results, after transduction into a wild strain, by the loss of activities specific to polynucleotide phosphorylase. A low phosphorolytic activity is nevertheless detectable in crude extracts, but no longer in extracts slightly purified after heat treatment at 54°C. The part played by other enzymes in these activities is discussed. Bacterial growth is not affected by introduction of the mutation.