Guido Volckaert

Locus-specific primers for LMW glutenin genes on each of the group 1 chromosomes of hexaploid wheat

To reveal the chromosomal location of three known low-molecular-weight (LMW) glutenin genes in wheat, we designed and used three sets of sequence-specific primers in polymerase chain reactions (PCR) on ‘Chinese Spring’ and its derived group 1 aneuploid nullisomic-tetrasomic stocks. Two sets proved to be chromosome specific and amplified sequences from the Glu-A3 and Glu-D3 loci, respectively. The third set was apparently composed of conserved sequences as it produced PCR products in each of the aneuploids. Two of these products were cloned, and their sequences differed from the known LMW glutenin genes at several positions. Again, primer sets specific for these sequences were designed. One set was directed to the Glu-A3 locus, the second set resulted in two PCR products differing in length, one of which was located on chromosome 1B and the other on 1D. Primer sets constructed for the latter two sequences were specific for the Glu-B3 and Glu-D3 loci, respectively. Hence, primer sets specific for each of the three homoeologous chromosomes of the group 1 (1A, 1B, 1D) are available. In addition, these locus-specific primers were assayed for their ability to distinguish among wheat cultivars. PCR products amplified with one of the Glu-A3-specific primer sets showed length polymorphisms in various wheat varieties. Varieties carrying the 1RS.1BL translocated chromosomes could be recognized by the absence of a PCR product when the Glu-B3 primer set was used. These results suggest that PCR with locus-specific primers can be useful in the molecular genetic analysis of hexaploid wheat.

Transient gene expression in electroporated banana (Musa spp., cv. ‘Bluggoe’, ABB group) protoplasts isolated from regenerable embryogenetic cell suspensions

SummaryElectroporation conditions were established for transient expression of introduced DNA in banana (Musa spp., cv. ‘Bluggoe’) protoplasts isolated from regenerable embryogenic cell suspensions. The following parameters were found to be highly influential: electroporation buffer, polyethylene glycol treatment and its duration before electroporation, use of a heat shock, and chimaeric gene constructs. The maximum frequency of DNA introduction as detected by an in situ assay for transient expression of the uidA gene, amounted to 1.8% of total protoplasts. Since plants have recently been regenerated from banana protoplasts at a high frequency, the present results may contribute to the production of transgenic banana.

Protection of turkeys against Chlamydia psittaci challenge by gene gun-based DNA immunizations

Particle-mediated (Helios Gene Gun) transfer to the turkey epidermis of plasmid DNA expressing the major outer membrane protein (MOMP) of an avian Chlamydia psittaci strain was evaluated for its ability to raise an immune response and protection against challenge with the homologous strain. In turkeys, the delivery of pcDNA1/MOMP coated onto 0.6 microm gold beads was the most efficient compared to immunisations using 1.0 or 1.6 microm gold beads. The delivery of as little as 1 microg pcDNA1/MOMP coated onto 0.6 microm gold beads was efficient. Immunisation with 1.0 microm gold beads required twice more (2 microg) DNA to achieve comparable results. The use of 2 microg DNA coated onto 1.6 microm gold beads had no effects. The gene gun delivery both primed T-helper and B-cell memory although recombinant MOMP-expressing cells did not induce high-titre antibody responses. The significance of gene gun-based DNA immunisation as a means of preventing severe clinical signs, lesions and chlamydia excretion in a turkey model of Chlamydia psittaci infection was demonstrated.

Use of a nested PCR-enzyme immunoassay with an internal control to detect Chlamydophila psittaci in turkeys

BackgroundLaboratory diagnosis of Chlamydophila psittaci, an important turkey respiratory pathogen, is difficult. To facilitate the diagnosis, a nested PCR-enzyme immunoassay (PCR-EIA) was developed to detect the Cp. psittaci outer membrane protein A (ompA) gene in pharyngeal swabs.MethodsThe fluorescein-biotin labelled PCR products were immobilized on streptavidin-coated microtiter plates and detected with anti-fluorescein peroxidase conjugate and a colorimetric substrate. An internal inhibition control was included to rule out the presence of inhibitors of DNA amplification. The diagnostic value of the ompA nested PCR-EIA in comparison to cell culture and a 16S-rRNA based nested PCR was assessed in pharyngeal turkey swabs from 10 different farms experiencing respiratory disease.ResultsThe sensitivity of the nested PCR-EIA was established at 0.1 infection forming units (IFU). Specificity was 100%. The ompA nested PCR-EIA was more sensitive than the 16S-rRNA based nested PCR and isolation, revealing 105 out of 200 (52.5%) positives against 13 and 74 for the latter two tests, respectively. Twenty-nine (23.8%) out of 122 ompA PCR-EIA negatives showed the presence of inhibitors of DNA amplification, although 27 of them became positive after diluting (1/10) the specimens in PCR buffer or after phenol-chloroform extraction and subsequent ethanol precipitation.ConclusionThe present study stresses the need for an internal control to confirm PCR true-negatives and demonstrates the high prevalence of chlamydiosis in Belgian turkeys and its potential zoonotic risk. The ompA nested PCR-EIA described here is a rapid, highly sensitive and specific diagnostic assay and will help to facilitate the diagnosis of Cp. psittaci infections in both poultry and man.

Protection of turkeys against Chlamydophila psittaci challenge by parenteral and mucosal inoculations and the effect of turkey interferon-gamma on genetic immunization.

Plasmid DNA (pcDNA1::MOMP A) expressing the major outer membrane protein of an avian Chlamydophila psittaci serovar A strain was tested for its ability to induce protective immunity against challenge with the same C. psittaci serovar. A combined parenteral (intramuscular injection) and mucosal route (DNA drops administered to the nares) of DNA inoculation was compared to three other, different routes of administration (intramuscular inoculation, DNA drops administered to the nares and aerosol immunization). In addition, the effect of turkey interferon gamma (tIFN‐γ) on intramuscular immunization was evaluated by co‐expressing pCIneo::tIFN‐γ. A significant level of protection was observed in turkeys immunized via the combined parenteral/mucosal route, the intramuscular route or by aerosol. Severe clinical signs and lesions were observed in the non‐vaccinated control groups, in 80% of turkeys inoculated with a mixture of pcDNA1::MOMP A and pCIneo::tIFN‐γ, and in 60% of turkeys vaccinated with DNA drops administered to the nares. The use of MOMP‐based DNA vaccination as a means of preventing severe clinical signs and lesions in a turkey model of C. psittaci infection was demonstrated, as was down‐regulation of the immune response by co‐expression of tIFN‐γ.

The lysis cassette of bacteriophage фKMV encodes a signal-arrest-release endolysin and a pinholin

The lysis cassette of Pseudomonas aeruginosa phage фKMV encodes a holin, endolysin, Rz and Rz1 in the canonical order. It has a tight organization with a high degree of overlapping genes and is highly conserved (between 96 and 100% identity at the protein level) among several other members of the “phiKMV-like viruses.” The endolysin KMV45 exhibits characteristics as expected for a signal-arrest-release (SAR) endolysin, whereas the holin KMV44 is a typical pinholin. KMV45 is initially secreted as an inactive, membrane-anchored endolysin, which is subsequently released by membrane depolarization driven by the pinholin KMV44. The SAR domain of KMV45 is necessary for its full enzymatic activity, suggesting a refolding of the catalytic cleft upon release from the membrane. The physical proximity of the catalytic glutamic acid residue close to SA R domain suggests an alternative activation mechanism compared to the SAR endolysin of phages P1, ERA103 and 21. Expression of KMV44 leads to a quick cell lysis when paired with SAR endolysin KMV45, but not with the cytoplasmic phage λ endolysin, indicating the membrane depolarizing function of KMV44 rather than the large hole-making function characteristic of classical holins.

Transient gene expression in transformed banana (Musa cv. Bluggoe) protoplasts and embryogenic cell suspensions

In order to introduce currently-available genes with agronomical value into banana, two genetic transformation protocols have been optimized.

Mechanism of Fluorescence and Conformational Changes of the Sarcoplasmic Calcium Binding Protein of the Sand Worm Nereis diversicolor upon Ca2+ or Mg2+ Binding

The calcium-binding protein isolated from the sarcoplasm of the muscles of the sand worm Nereis diversicolor has four EF-hands and three active binding sites for Ca(2+) or Mg(2+). Nereis diversicolor sarcoplasmic calcium-binding protein contains three tryptophan residues at positions 4, 57, and 170, respectively. The Wt protein shows a very limited fluorescence increase upon binding of Ca(2+) or Mg(2+). Single-tryptophan-containing mutants were produced and purified. The fluorescence titrations of these mutants show a limited decrease of the affinity for calcium, but no alterations of the cooperativity. Upon adding calcium, Trp170 shows a strong fluorescence increase, Trp57 an extensive fluorescence decrease, and Trp4 shows no fluorescence change. Therefore mutant W4F/W170F is ideally suited to analyze the fluorescence titrations and to study the binding mechanism. Mutations of the calcium ligands at the z-position in the three binding sites show no effect at site I and a total loss of cooperativity at sites III and IV. The quenching of Trp57 upon calcium binding is dependent on the presence of arginine R25, but this residue is not just a simple dynamic quencher. The role of the salt bridge R25-D58 is also investigated.

A general method for routine sequencing of cloned DNA fragments using commercial dye primers and its application in sequence analysis of Toxoplasma gondii RH genome fragments

A generally applicable approach for amplification and subsequent sequencing using commercially available dye primers is described. The polymerase chain reaction primers, one of which is biotinylated, are tagged at their 5 end with sequences of commercial sequencing primers. After purification and strand separation on streptavidin-coated magnetic beads, both strands are sequenced automatically on an Applied Biosystems 373A DNA sequencer using the appropriate standard dye-labeled sequencing primers. This approach is demonstrated on PstI and MnlI DNA fragments from Toxoplasma gondii RH cloned in the in-house developed derivatives of vector pJRtac99.

Regulation of inhibin α- and βA-subunit messenger ribonucleic acid levels by gonadotropins and IGF-I in cultured chicken granulosa cells

Abstract A quantitative competitive reverse transcription polymerase chain reaction assay (QC RT-PCR) for quantifying the absolute levels of the expression of inhibin α- and β A -subunits in chicken granulosa cells showed that these subunits are expressed in different amounts depending on follicular maturation. The present study determined the regulation of the expression of these subunits. The individual effect of different doses of IGF-I, LH or FSH (1–100xa0ng/ml) or the combination of IGF-I with either LH or FSH at different concentrations, on the expression of inhibin α- and β A -subunit was determined on cultured granulosa cells of F 1 and the combined F 4 +F 5 follicle. Cells were cultured for 48xa0h in 6-well plates with or without added hormones. Culture medium was discarded, cells were washed and total RNA was extracted from the cells. Five hundred nanograms of total RNA was reverse transcribed using specific primers and coamplified with an internal standard, as described previously, to determine expression level in the cells. IGF-I, LH, and FSH enhanced the inhibin α-subunit mRNA levels in a dose dependent manner in both F 1 and the combined F 4 +F 5 whereas inhibin β A -subunit was not affected. The effects of FSH, LH were more expressed in F 1 follicles compared to F 4 +F 5 on the α-subunit. The addition of IGF-I and either LH or FSH during the culture period significantly increased the stimulatory effects of both LH and FSH on the expression of inhibin α-subunit in F 1 follicles but had no significant effect on the inhibin β A -subunit. The results suggest that the changing expression levels of inhibin α-subunit during follicular development are the result of the regulatory effect of the interaction between IGF-I and the gonadotropins and that the regulation of this subunit may be the main factor for the regulation of the protein inhibin levels. Other factors may be also implicated in the changing expression levels of the β A -subunit.