Albert Herzog

Specific identification of Bordetella pertussis by the polymerase chain reaction

Oligonucleotide primers were used to amplify specific DNA regions of the Bordetella pertussis genome by the polymerase chain reaction. One pair of primers, PTp1/PTp2, identified a 191-bp DNA fragment located in the regulatory region of the pertussis toxin operon; a second pair of primers led to amplification of a 121-bp DNA piece located in an insertion-like element specific to B. pertussis. Both sets of primers were able to discriminate between the pathogen and related Bordetella species; they detected down to 6 bacteria and appeared suitable for routine detection of B. pertussis in clinical specimens.

Alteration of ribosomal protein S17 by mutation linked to neamine resistance in Escherichia coli: I. General properties of neaA mutants

A mutant of Escherichia coli strain K12S isolated after selection for resistance to the amino glycoside antibiotic neamine shows severe restriction of amber suppressors in vivo . Ribosomes from the mutant exhibit low neamine-induced misreading in vitro and a decreased affinity for the related antibiotic streptomycin. The S17 ribosomal protein from the mutant strain shows altered electrophoretic mobility on two-dimensional polyacrylamide gels and also differs in chromatographical behaviour from the S17 protein from the parental strain. When ribosomes from the mutant strain are reconstituted in the presence of a molar excess of the S17 protein from the wild type species, the particles exhibit normal druginduced misreading properties as well as a normal capacity for binding streptomycin. The gene involved in the alteration of protein S17 ( rpxQ ) is located inside the ribosomal protein gene cluster at 64 minutes on the E. coli genetic map and cotransduces at 96% with rpxE (S5).

Detection of Borrelia burgdorferi in biological samples using the polymerase chain reaction assay

Oligonucleotide primers were used in the polymerase chain reaction assay to amplify specific DNA regions of the Borrelia burgdorferi 49-kb linear plasmid. One set of primers identifies a 442-bp DNA fragment in the OspA gene and a second pair of amplimers, a 176-bp DNA piece located in the OspB gene. The last set of primers, OspBpc3/pc4, outperformed the other pair in discriminating pathogenic North American or European isolates from related bacterial species, detected down to 4 spirochaetes, and was suitable for the identification of B. burgdorferi in biological samples, such as synovial and cerebrospinal fluids.

Cooperative control of translational fidelity by ribosomal proteins in Escherichia coli

SummaryThe effect on translational fidelity of a particular mutation in the gene coding for protein S5 (rpxE) has been investigated. This mutation has the opposite effect of a restrictive strA mutation; in vivo, it relieves the restriction imposed by strA on the suppression of T4 nonsense mutants and results in hypersensitivity to streptomycin; in vitro, the presence of the altered S5 protein in 30S ribosomes results in increased intrinsic misreading. It is concluded that this mutation, ramC319, acts as a ribosomal ambiguity mutation similar to certain mutations of protein S4 (ramA).

Molecular cloning of human haptoglobin cDNA: Evidence for a single mRNA coding for alpha 2 and beta chains:

Human haptoglobin (Hp) is a plasma glycoprotein composed of alpha and beta polypeptide chains that are covalently associated by disulfide bonds. It had been suggested that alpha and beta polypeptides could be synthesized via a common precursor polypeptide. We report the molecular cloning of DNA complementary to human Hp mRNA. One of the clones, pULB1148, carries a full length copy coding for both alpha 2 and beta polypeptides. In vitro translation of human liver mRNA hybridizing with this cDNA gives a protein mol. wt. of 49000 daltons. The sequence of the alpha 2 beta cDNA shows the presence of a single Arg residue between Gln 142 of the alpha 2 chain and Ileu 1 of the beta chain. With a few minor exceptions, the DNA sequence fits the previously published amino acid sequences. The differences are the presence of an Asp residue at position 52 of alpha 2 instead of Asn, the existence in beta of only one Lys residue between Gly 65 and the following Gln, the presence of Ser and Cys at positions 218‐219 instead of Cys‐Ser, and of Asp residues at positions 205 and 235 instead of Asn.

Fluorescence studies on the 30 S ribosome assembly process

E. coli 30 S ribosomal subunits may be reconstituted in vitro from their protein constituents and 16 S RNA [l] . The complexity of the assembly process was clarified to a large extent by Mizushima and Nomura in a recent publication [2] where the authors proposed an assembly map for the 30 S ribosome. We were also concerned with the assembly process and chose as experimental principle to introduce a fluorescent marker (ethidium bromide) in the reconstitution system and to follow the evolution of the fluorescence intensity during the reconstitution process. Our results indicate that some base-paired regions of the 16 S RNA molecule are involved in the assembly process and that the recognition of these regions by 30 S ribosomal proteins is highly specific. Moreover, it appears that only a few ribosomal proteins interact with the 16 S RNA base-paired regions and that an intermediate complex forms in the presence of ethidium bromide. This complex has a sedimentation coefficient of about 20 S and a protein composition similar to that one of the ribosomal subunit precursor 21 S accumulating in a cold sensitive mutant [3] . This provides strong correlation between in vivo and in vitro results and supports the relevance of in v&o assembly studies to the biological process. 16 S RNA, total, core and split proteins from 30 S ribosomes were prepared as described in Traub and Nomura paper [l] . Pure ribosomal proteins were isolated on carboxymethyl cellulose columns [4] and electrophoresis performed in 8 M urea gels, pH 4.4, acrylamide 7.5% and bisacrylamide 0.8%. 30 S ribosomes reconstitution was followed by the fluorescence variation of ethidium bromide charged

Characterization of human haptoglobin cDNAs coding for α2FSβ and α1Sβ variants

A human liver library, derived from a heterozygous (Hp2‐1) donor, has been used to isolate cDNA clones coding for the haptoglobin (Hp) α1Sβ and α2FSβ variants. DNA sequencing has shown that the two variants are identical except for the αF duplicated segment in Hp α2FSβ. Four nucleotide changes have been found between the phenotypically different F and S regions of the Hp α2 gene, resulting in an Asp,Lys/Asn,Glu substitution.

The ribosomal protein altered in spectinomycin resistant Escherichia coli.

In a previous paper [l] using reconstitution experiments 121 we reported the identification of the 30 S ribosomal protein conferring sensitivity to the antibiotic spectinomycin in Escherichiu coli. The present work shows that the mutation to spectinomycin resistance (spc’) significantly alters one particular protein of the 30 S moeity. This modification is revealed by chromatographic analysis of several independent spc’ mutants.

AN EFFECT OF STREPTOMYCIN ON THE DISSOCIATION OF ESCHERICHIA COLI 70 S RIBOSOMES.

Abstract It has been suggested by Spotts and Stanier (1961) that the primary site of action of streptomycin (Sm) is the ribosomes. According to their hypothesis, the different alleles at the streptomycin locus would direct the synthesis of structurally different ribosomes, and this would be responsible for sensitivity (Sms), resistance (Smr) or dependence (Smd) towards the antibiotic. This hypothesis was reinforced by reports from several laboratories ( Flaks et al., 1962 ; Mager et al., 1962 ; Speyer et al., 1962 ) that in cell-free amino acids incorporating systems, streptomycin exerts a strong inhibitory effect, when the ribosomal fraction has been purified from a Sms strain, but that it has only a small effect when the ribosomes originate from Smr or Smd bacteria. The aim of this work was to look for a possible effect of streptomycin on the centrifugation patterns of ribosomes, using a Sms and a Smr strain of E. coli .

Expression of human α1-antitrypsin in Escherichia coli

Complementary DNA coding for human α1‐antitrypsin has been placed under the control of the λPR promotor carrier by the expression vector pCQV2 [1]. In conditions which allow transcription from this promotor (thermoinactivation of the repressor), Escherichia coli cells harbouring the recombinant plasmid pULB1114 express human α1‐antitrypsin (± 9000 molecules/cell). The product has a M r of 44 000, corresponding to mature unglycosylated α1‐antitrypsin.