Alex Bollen

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.

Ixodes ticks belonging to the Ixodes ricinus complex encode a family of anticomplement proteins.

The alternative pathway of complement is an important innate defence against pathogens including ticks. This component of the immune system has selected for pathogens that have evolved countermeasures. Recently, a salivary protein able to inhibit the alternative pathway was cloned from the American tick Ixodes scapularis (Valenzuela et al., 2000; J. Biol. Chem. 275, 18717–18723). Here, we isolated two different sequences, similar to Isac, from the transcriptome of I. ricinus salivary glands. Expression of these sequences revealed that they both encode secreted proteins able to inhibit the complement alternative pathway. These proteins, called I. ricinus anticomplement (IRAC) protein I and II, are coexpressed constitutively in I. ricinus salivary glands and are upregulated during blood feeding. Also, we demonstrated that they are the products of different genes and not of alleles of the same locus. Finally, phylogenetic analyses demonstrate that ticks belonging to the Ixodes ricinus complex encode a family of relatively small anticomplement molecules undergoing diversification by positive Darwinian selection.

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.

The structural gene for the ribosomal protein S18 in Escherichia coli. I. Genetic studies on a mutant having an alteration in the protein S18

Abstract A mutant of Escherichia coli strain CR341, originally isolated as a temperature-sensitive mutant, was found to have an altered 30 S ribosomal protein (S18) in addition to and independently of temperature sensitivity. Protein S18 from the mutant strain differs in electrophoretic mobility in polyacrylamide gel electrophoresis at pH 4.5 from protein S18 of the parental origin. The mutation responsible for the alteration in S18 is different from two other mutations in the mutant strain which give the temperature-sensitive phenotype. The gene involved in the S18 alteration is located in a region between 76 and 88 minutes on the E. coli genetic map; the location is outside the str-spc region at 64 minutes, where several known ribosomal protein genes are located. An episome covering the loci rha (76 min) through pyr B (84 min) was introduced into the mutant. The resultant merodiploid strains were shown to produce both the normal and the mutant forms of S18. The results support the conclusion described in the accompanying paper (Kahan et al ., 1973) that the mutation studied is in the structural gene for S18.

Engineering of non-conventional yeasts for efficient synthesis of macromolecules: the methylotrophic genera

Methylotrophic yeasts, named after their ability to grow on methanol as the sole carbon source, have raised large interest as recombinant protein factories. In this review, we explain the basic mechanisms underlying this interest and describe the minimal requirements to transform the two genera recognized as methylotrophic, Pichia and Candida, into a powerful protein production tool. We present a comparison between this group of yeasts and the conventional yeasts used as expression system in view of productivity, level of secretion and quality of post-translational modifications. Selected examples of recombinant protein produced by methylotrophic yeast are also included.

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

The structural gene for the ribosomal protein S18 in Escherichia coli: II. Chemical studies on the protein S18 having an altered electrophoretic mobility

Abstract A mutant of Escherichia coli strain CR341 has an altered 30 S ribosomal protein S18. The alteration involves a change in the electrophoretic mobility of S18. S18 proteins were purified from the mutant and the parent strain, respectively, and their amino acid composition and tryptic peptides were compared. The results have shown that the mutational alteration involves substitution of cysteine for arginine. In addition, we determined the electrophoretic mobility of S18 proteins modified by ethyleneimine. The modification, which involves conversion of cysteine residues to S-(2-aminoethyl)cysteine, causes a greater electrophoretic mobility increase in the mutant protein than in the wild type protein, resulting in identical mobilities for the aminoethylated proteins. This experiment gives further support to the conclusion that the original mobility difference between mutant and wild type proteins is due to the mutational substitution of cysteine for arginine. The S18 obtained from a recombinant was also studied. The recombinant protein was found to have the mobility of the wild type protein and the wild type primary structure, as judged by amino acid composition and tryptic peptide analysis. This recombinant was obtained from the mutant by introducing Hfr strain G10 chromosome segments in the region between 70 and 10 minutes, and not in the str-spc region at 64 minutes, as described in the preceding paper. These results, together with those in the preceding paper, show that the mutation studied here is in the structural gene for S18, and that it maps outside the str-spc region.

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.

A PCR based DNA hybridisation capture system for the detection of human cytomegalovirus. A comparative study with other identification methods.

A simple, sensitive and specific colourimetric hybridisation method for the detection of HCMV DNA in clinical specimens is described. This method combines a PCR assay with a sensitive sandwich hybridisation assay. It relies on the use of a specific capture probe linked covalently to polystyrene microplates and a specific polybiotinylated detection probe. Amplified DNA fragments, sandwiched between these two probes, are detected by an enzymatic colour reaction. This PCR-based colourimetric hybridisation method was compared with other known HCMV detection methods. Clinical specimens (n = 145, corresponding to 106 patients) were tested by both a nested PCR assay and this colourimetric hybridisation method; and by either the culture method or the pp65 antigenaemia test depending on the type of sample used. The results showed that the PCR-based hybridisation method has a specificity similar to tissue culture, known as the conventional gold standard method, and could be used for the examination of the clinical specimens.

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.