Sequence and level of endogenous expression of calcium channel β subunits in Schistosoma mansoni displaying different susceptibilities to praziquantel

Kohn et al. [J. Biol. Chem. 276 (2001) 36873] demonstrated that cells expressing the structurally unusual schistosome β subunit SmCa_vβ1 in their voltage-operated calcium channels, exhibit an increased current amplitude in the presence of praziquantel (PZQ). This suggests that the beta subunit is involved in PZQ activity and is consistent with the known pharmacological effects of the drug. If this is so, the low susceptibility to PZQ noted in some Schistosoma mansoni strains could be due to some mutation(s) in the gene coding for this protein. We have sequenced the cDNAs coding for the SmCa_vβ1 and SmCa_vβ2 subunits of different sensitive and resistant strains and we have not been able to detect any meaningful differences. As an alternative hypothesis, the different sensitivity of schistosomes to PZQ action could be due to the expression of different β subunits in the parasite. This interpretation could also explain the low PZQ susceptibility of immature worms (28 days). We analyzed Northern blots of various strains and various developmental stages, but we were unable to demonstrate major quantitative differences in the expression of the β subunits.     

DNA sequence analysis of an allelic variant of the Actinobacillus pleuropneumoniae-RTX-toxin I (ApxIA) from serotype 10

The structural gene encoding Actinobacillus pleuropneumoniae-RTX toxin I (ApxIA), one of the major hemolytic and cytolytic toxins of the organism, was cloned from serotype 10. The nucleotide sequence data showed that the gene from serotype 10 was 98% identical to that from serotype 1 at both DNA and amino acid levels. The sequence difference was found to localize at the 3′ terminal region of the gene. We then analyzed the 3′ terminal region of the apxIA gene from other serotypes, 5a, 5b, 9 and 11, using polymerase chain reaction-amplified DNA fragments. Results of DNA sequence indicated that apxIA gene can be divided into the original form including serotypes 1, 9 and 11, and the allelic variant including serotypes 5a, 5b and 10. These gene products, ApxIA proteins, appear to have different second structures at the carboxyl terminal proximal region.     

Relevance of IL-1 and TNF gene polymorphisms on interleukin-1β and tumor necrosis factor-α gastric mucosal production

We aimed to evaluate the influence of Helicobacter pylori infection and IL-1/TNF gene polymorphisms on interleukin (IL)-1β and tumor necrosis factor (TNF)-α gastric mucosal production. IL-1β and TNF-α levels in homogenized biopsy specimens taken from the antrum and corpus of 81 patients were measured by enzyme-linked immunosorbent assay. Genomic DNA was typed for the IL1B-511, IL1B+3954, variable number of tandem repeat (VNTR) IL1RN, TNFA-308, TNFA-238, LTA NcoI, and LTA Bsi gene polymorphisms by polymerase chain reaction, restriction fragment length polymorphism, and TaqMan assays. H. pylori infection and CagA/VacA antibody status were determined by Western blot. IL-1β and TNF-α protein levels were significantly higher in the gastric antrum of patients infected with H. pylori compared with uninfected patients [9.54 (5.07–16.28) vs. 4.55 (3.69–8.28) pg IL-1β/mg protein, p = 0.004, and 1.5 (0.7–2.71) vs. 0.63 (0.3–1.26) pg TNF-α/mg protein, p = 0.001]. Among H. pylori-infected individuals, carriers of the IL1RN*2 allele had significantly higher antrum mucosal IL-1β levels than noncarriers [15.97 (9.59–26.6) vs. 10.08 (7.72–13.33), p = 0.008]. No association between gastric mucosal TNF-α levels and genotypes of the TNFA and LTA gene polymorphisms was reported. Our results indicate that the VNTR polymorphism of the IL1RN gene influences IL-1β gastric mucosal production in patients infected with H. pylori.     

Genotypic characterization of enteropathogenic Escherichia coli (EPEC) isolated in Belgium from dogs and cats

Enteropathogenic Escherichia coli (EPEC) are isolated from man and farm animals but also from dogs and cats. They produce typical histological lesions called ‘attaching and effacing’ lesions. Both plasmid and chromosomal elements are involved in the pathogenesis of EPEC infection. The presence of these genetic elements was investigated in 14 dog and three cat EPEC isolates. A bfpA-related gene was detected in five of the 17 isolates in association with high molecular weight plasmids, and a locus of enterocyte effacement (LEE) was present in all isolates. The LEE was inserted in the selC region in only 12% of the isolates. The eae, tir, espA and espB genes were analyzed by multiplex PCR. The results indicated the presence of those genes in the tested isolates with heterogeneity in the gene subtypes present: eaeγ-tirα-espAα-espBα (65%), eaeβ-tirβ-espAβ-espBβ (29%), eaeα-tirα-espAα-espBα (6%). Moreover, the espD gene was also present in dog and cat EPEC. The DEPEC and CEPEC form a heterogeneous group and five of them are closely related to human EPEC.     

Identification of an upstream activation site in the pyruvate decarboxylase structural gene (PDC1) of Saccharomyces cerevisiae

Summary The upstream region of the Saccharomyces cerevisiae pyruvate decarboxylase structural gene, PDC1, has been isolated and fused to the indicator gene Escherichia coli lacZ. 1.2 kb of the upstream region has been sequenced. The PDC1-lacZ fusion has been integrated at the ura3-52 locus in the yeast genome, and has a basal level of expression on ethanol. On glucose media this level is increased 30–50 fold. An upstream activation site, UAS_pdc, between 793 and 535 by upstream from the ATG of PDC1, which mediates the response to glucose has been identified by deletion analysis. The UAS_pdc contains a consensus RPG box, originally identified in ribosomal protein genes (Leer et al. 1985). The function of UAS_pdc is independent of distance from the ATG. There is also an upstream repressing sequence located between 535 and 385 by upstream from the translational start of PDC1.     

Characterization of a Chlamydia psittaci DNA Binding Protein (EUO) Synthesized during the Early and Middle Phases of the Developmental Cycle

The EUO gene (for early upstream open reading frame) of Chlamydia psittaci was previously found to be transcribed better at 1 than at 24 h postinfection. We found that the EUO gene encodes a minor protein that is expressed within 1 h of infection of host cells with C. psittaci 6BC but that protein quantity peaks during the logarithmic growth phase of reticulate bodies (RBs), declines late in the infection (after 20 h) when RBs reorganize into elementary bodies (EBs), and is absent in infectious EBs. EUO protein lacks homology to known proteins but does contain a putative helix-turn-helix motif. We found that recombinant EUO binds to DNA in vitro with a relatively broad specificity. Using the bp −200 to +67 promoter region of the cysteine-rich envelope protein (crp) operon as a model, we show that EUO protein preferentially binds to AT-rich sequences and protects crp DNA from DNase I from approximately bp −60 to −9. We also found that native EUO protein in extracts of RBs binds to the promoter region of the crp operon, demonstrating that the DNA binding property of EUO protein is not an artifact of recombinant methods. Although EUO protein appears to bind to the crp operon with high affinity in vitro (K_d of about 15 nM), it is not known whether the protein binds the crp DNA in vivo.     

5′-UTR Structural Organization, Transcript Expression, and Mutational Analysis of the Human Rab Geranylgeranyl Transferase α-Subunit (RABGGTA) Gene

Hermansky-Pudlak syndrome (HPS) is a recessively inherited disease with dysfunction of several related subcellular organelles including platelet-dense granules, melanosomes, and lysosomes. Our recent identification of the mutation in murine Rab geranylgeranyl transferase α-subunit gene (Rabggta) in one mouse model of HPS, the gunmetal mouse, suggested that human patients with similar phenotypes might have mutations in the human orthologous RABGGTA gene. This prompted reanalysis of the 5′-untranslated structure of the human RABGGTA gene in normal individuals and in patients with deficiencies of platelet-dense granules (αδ-SPD), alpha granules (α-SPD or gray platelet syndrome, GPS) or alpha plus dense granules (αδ-SPD). We report the complete sequence of intron α of RABGGTA and demonstrate that exon α is immediately upstream of intron α. The exon/intron structural organization of the 5′-untranslated region (UTR) of human RABGGTA was found to be similar to that of the mouse Rabggta gene. However, exons α and introns α are not homologous between mouse and human. Features of the 5′-UTR of RABGGTA suggest it is a housekeeping gene. While obvious disease-causing mutations of human RABGGTA were not found in our existing SPD patients by sequencing its entire coding region, several polymorphisms of RABGGTA including a putative cryptic splicing mutation in intron 4 were identified. Knowledge of the 5′-UTR structure of RABGGTA and its common polymorphisms will be useful for mutation screening or linkage analysis in patients with albinism, thrombocytopenia, or platelet SPD.     

The MAL63 gene of Saccharomyces encodes a cysteine-zinc finger protein

Summary Inducible maltose fermentation by tSaccharomyces carlesbergensis requires the product of the MAL63 gene of the MAL6 locus. It has been suggested that this gene product is an activator protein controlling the expression of the structural genes encoding the maltose fermentative enzymes perhaps by binding to DNA sequences upstream of these genes. We report the sequence of the MAL63 gene. A single open reading frame is seen capable of encoding a protein of 470 amino acid residues. The deduced sequence of this protein indicates that it is a cysteine-zinc forger protein supporting the hypothesis that the MAL63 gene product is a DNA binding protein.