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Biomedical subjects

D G Wild

Publications and source records attributed to D G Wild.

At least 19 recordsLinked to original sources
PubMed1997

The effects of mutations in the rpmB,G operon of Escherichia coli on ribosome assembly and ribosomal protein synthesis.

The rpmB,G operon of Escherichia coli codes for proteins L28 and L33 of the larger (50S) ribosomal subunit. Strains with mutations in this operon can help define the roles of these proteins in ribosome synthesis and function. One such strain, BM108, makes neither protein and is unable to synthesize completed ribosomes; instead ribonucleoproteins accumulate, in the form of '30S material' and '47S particles'. However, when protein L28 is supplied from a plasmid, the growth rate, the kinetics of ribosome synthesis and the coordination of ribosomal protein synthesis are no different from that in wild-type organisms even though protein L33 is missing. This suggests that the latter protein can be redundant for ribosome synthesis and function. Another mutant strain, BM81, has a frameshift mutation that gives rise to an oversized protein L28. This mutant accumulates 30S material and 47S particles during slow exponential growth. The composition of the 47S particles from strains BM81, BM108 and a third mutant strain, TP28, suggests that their defining feature is the absence of L28; this is further evidence for an important role for this protein in ribosome assembly. Accumulation of ribonucleoproteins in strains BM81 and BM108 leads to some loss of the ordinarily precise coordination of synthesis of ribosomal proteins. We describe and discuss the characteristic features of this unbalanced synthesis.

Aniline Compounds↗
PubMed1997

The roles of proteins L28 and L33 in the assembly and function of Escherichia coli ribosomes in vivo.

Strain BM108 of Escherichia coli has a chromosomal mutation in the rpmB,G operon that prevents synthesis of ribosomal proteins L28 and L33. The mutation was lethal unless synthesis of protein L28 was induced from a plasmid. Without protein L28, RNA and protein synthesis were linear rather than exponential. No 70S ribosomes were made. Instead, RNA accumulated in '30S material' and '47S particles'; the latter were distinct from 50S ribosomal subunits, lacked proteins L28 and L33 and had substoicheometric amounts of three other proteins. When L28 synthesis was induced (but protein L33 was still absent), the strain grew as well as, and assembled 70S ribosomes with similar kinetics to, a wild-type control. Thus, protein L28 is required for ribosome assembly in strain BM108 while protein L33 has no significant effect on ribosome synthesis or function.

Aniline Compounds↗
PubMed1997

Mutations in the rpmBG operon of Escherichia coli that affect ribosome assembly.

The rpmBG operon of Escherichia coli codes for ribosomal proteins L28 and L33. Two strains with mutations in the operon are AM81, whose ribosomes lack protein L28, and AM90, whose ribosomes are without protein L33. Neither strain showed major defects in ribosome assembly. However, when the mutations were transferred to other strains of E. coli, ribosome synthesis was greatly perturbed and precursor ribonucleoproteins accumulated. In the new backgrounds, the mutation in rpmB was complemented by synthesis of protein L28 from a plasmid; the rpmG mutation was not complemented by protein L33 because synthesis of protein L28 from the upstream rpmB gene was also greatly reduced. The results suggest that protein L33, in contrast to protein L28, has at best a minor role in ribosome assembly and function.

Amino Acid Sequence↗
PubMed1993

Ribosome assembly in three strains of Escherichia coli with mutations in the rpmB,G operon.

The rpmB,G operon of Escherichia coli codes for the synthesis of ribosomal proteins L28 and L33. In one mutant strain (TP28), these two proteins are made at about half their normal rates, ribosome assembly is greatly perturbed and precursor particles accumulate. The mutation in strain TP28 is in a Shine-Dalgarno sequence in the leader region of the rpmB,G messenger RNA. Another mutant, strain AM108, makes neither protein because it has the mobile element IS1 inserted into the rpmB coding sequence. Surprisingly, ribosome assembly in this strain is virtually normal with respect to growth rate. Strain AM90, which fails to make protein L33, has the element IS3 inserted into rpmG and also shows no major defects in ribosome assembly.

Amino Acid Sequence↗
PubMed1989

Some properties of two erythromycin-dependent strains of Escherichia coli.

Strains of Escherichia coli can be isolated that require erythromycin for growth. With one strain, AM, a range of antibiotics, including chloramphenicol, tetracycline, spectinomycin, kasugamycin and rifampicin, will substitute for erythromycin on solid and in liquid media; nalidixic acid supports growth in liquid but not on solid media. With a second strain, 103, chloramphenicol, tetracycline and spectinomycin support growth in liquid media but on solid medium only chloramphenicol substitutes for erythromycin. In media of higher than normal ionic strength, strain AM, but not strain 103, can grow in the absence of antibiotics. Possible reasons for these complex phenotypes are discussed.

Aminoglycosides↗
PubMed1988

Reversion from erythromycin dependence in Escherichia coli: strains altered in ribosomal sub-unit association and ribosome assembly.

A mutant of Escherichia coli dependent on erythromycin for growth spontaneously gives erythromycin-independent strains with altered or missing ribosomal proteins. strains with defects in ribosome assembly were sought and obtained from among these revertants. Two organisms in which ribosomal protein L19 is altered and absent respectively have 70S ribosomes whose dissociation into sub-units is particularly sensitive to pressures generated during centrifuging. The mutant that lacks protein L19 also accumulates ribosome precursor particles during exponential growth as do others including mutants that lack proteins S20 or L1. These strains also show unbalanced synthesis of RNA and so will be useful in investigating both the pathways and the regulation of ribosome assembly.

Bacterial Proteins↗
PubMed1985

The location of a mutation affecting ribosomal protein synthesis by Escherichia coli.

A mutation in a strain of Escherichia coli 15 produced ribosomes by an abnormal pathway that caused the accumulation of 47S ribonucleoprotein particles. The mutation was transferred to strains of E. coli K12 by transductions with bacteriophage P1cam and was at about 82 min, between cysE and pyrE and rather closer to the latter. The location and the physiological properties of the mutant suggested that the mutation was in the rpmB,G transcription unit and affected the synthesis of ribosomal proteins L28 and L33.

Centrifugation, Density Gradient↗
PubMed1985

Interactions between mutations affecting ribosome synthesis in Escherichia coli.

RNA synthesis was followed during amino acid starvation of strains of Escherichia coli that contained both the relaxed (relA) mutation and a mutation affecting ribosome assembly that results in oversynthesis of RNA. The ribosome mutation did not by itself lead to relaxedness. The relaxed mutation could be expressed in organisms that contained the ribosome mutation.

Amino Acids↗
PubMed1984

Ribosomal protein synthesis by a mutant of Escherichia coli.

The mutant strain of Escherichia coli, TP28, synthesises ribosomes by an abnormal pathway and accumulates large quantities of 47S ribonucleoprotein particles. The protein complement of mutant 70S ribosomes is normal but 47S particles contain only traces of proteins L28 and L33 and have a significantly reduced content of four other proteins. The mutation reduces the rates of synthesis of L28 and L33 by about half but other widespread alterations ensue. In particular, ribosomal protein synthesis in the mutant strain becomes less well balanced than in its parent: some proteins, particularly those from promoter-proximal genes, are oversynthesized and their excess then degraded.

Escherichia coli↗
PubMed1982

Effects of iodoamphenicol on ribosome assembly in two strains of Escherichia coli.

When the growth of Escherichia coli strain 15TP was inhibited by iodoamphenicol, three 'iodoamphenicol particles' accumulated with sedimentation coefficients of 25S, 33S and 45S. The 25S and 33S particles differ in sedimentation properties from equivalent ribosome precursor particles detected during pulse-labelling of exponentially growing cells. Inhibition of a mutant, strain 15-28 (defective in ribosome assembly), by iodoamphenicol resulted in the accumulation of 38S iodoamphenicol particles that are different from the particles made by the parent. The results support the contention that assembly of 50S ribosomal subunits by the mutant is altered at an early stage.

Bacterial Proteins↗
PubMed1981

Resistance to fluorouracil in Candida utilis: effects on the uptake of pyrimidines and amino acids.

5-Fluorouracil powerfully inhibits growth of Candida utilis. Isolates that are resistant to fluorouracil all have a reduced ability to transport uracil but most also have other defects. Their capacity to take up a wide range of amino acids is greatly reduced, as is their ability to alter rates of amino acid transport during nitrogen starvation. These isolates may be defective in the coupling of energy generation to transport systems.

Amino Acids↗
PubMed1980

Intermediates in the assembly of ribosomes by a mutant of Escherichia coli.

Escherichia coli strain 15--28 is a mutant that accumulates ribonucleoprotein ('47 S') particles during exponential growth. These particles contain mature 23 S rRNA, but lack three of the proteins of the larger ribosomal subunit, to which they are a precursor. In organisms growing at 20 degrees C, assembly of 47 S particles involves three intermediates that contain precursor 23 S rRNA, one of which has the same sedimentation properties as 47 S particles. Assembly of 50 S ribosomal subunits in the parent strain is 'normal'. There are three intermediates; each contains precursor 23 S rRNA, and one cannot be distinguished from completed subunits by sedimentation. Synthesis of 30 S ribosomal subunits in parent and mutant strains is qualitatively similar, but quantitatively different. When growth is at 37 degrees C, assembly in the mutant alters. There are now two sequential precursors to 47 S particles. Both contain precursor 23 S rRNA; one has the same sedimentation coefficient as 47 S particles. In some respects, synthesis in the mutant proceeds as though 47 S particles, rather than 50 S ribosomal subunits, are the end-product of assembly.

Centrifugation, Isopycnic↗
PubMed1979

Abnormal ribosome assembly in a mutant of Escherichia coli.

The mutant strain, 15--28, of Escherichia coli accumulates ribonucleoprotein ('47S') particles that were previously shown [Markey, Sims & Wild (1976) Biochem. J. 158, 451--456] to be an unusual intermediate in the assembly of 50S ribosomal subunits...

Centrifugation, Density Gradient↗
PubMed1978

Binding of chloramphenicol and a fragment of aminoacyl-transfer ribonucleic acid to ribosomes and a ribosome precursor from a mutant of Escherichia coli.

During exponential growth, the mutatn strain Escherichia coli 15-28 accumulates 47S particles, which are unusual precursors to 50S ribosomal subunits. The 47S particles have little ability to bind chloramphenicol, but binding of a fragment of aminoacyl-tRNA is about half that by completed subunits. The 70S (and 50S) ribosomes of strain 15-28 and its parent (strain 15TP) do not differ in chloramphenicol binding. Although ribosomes from the mutant are less able than those from the parent to bind the fragment, this difference is not as marked as was found previously [Sims & Wild (1976) Biochem. J. 160, 721-726] for the binding of an analogue of peptidyl-tRNA and for peptidyltransferase activity. The altered activities may arise because strain 15-28 misassembles 50S subunits of altered conformation and because the few proteins that 47S patricles lack have vital functions in some of the partial reactions of protein synthesis.

Binding Sites↗