Genetic control of PI and GC variants in the American Mink

Genetic polymorphism of the serum α-protease inhibitor (PI) and group-specific component (GC) in minks was revealed using one-dimensional polyacrylamide gel electrophoresis and immunoblotting. Two codominant alleles were identified at each of the two loci. The data ruled out the possibility of any linkage between the PI, GC and the coat colour gene Crystal ( Cr ).     

A PCR-based method for diet analysis in freshwater organisms using 18S rDNA barcoding on faeces

The development of DNA barcoding from faeces represents a promising method for animal diet analysis. However, current studies mainly rely on prior knowledge of prey diversity for a specific predator rather than on a range of its potential prey species. Considering that the feeding behaviour of teleosts may evolve with their environment, it could prove difficult to establish an exhaustive listing of their prey. In this article, we extend the DNA barcoding approach to diet analysis to allow the inclusion of a wide taxonomic range of potential prey items. Thirty-four ecological clade-specific primer sets were designed to cover a large proportion of prey species found in European river ecosystems. Selected primers sets were tested on isolated animal, algal or plant tissues and thereafter on fish faeces using nested PCR to increase DNA detection sensitivity. The PCR products were sequenced and analysed to confirm the identity of the taxa and to validate the method. The methodology developed here was applied to a diet analysis of three freshwater cyprinid species that are assumed to have similar feeding behaviour [Chondrostoma toxostoma toxostoma (Vallot 1837), Chondrostoma nasus nasus (Linnaeus, 1758) and Barbus barbus, (Linneaus 1758)]. These three species were sampled in four different hydrographic basins. Principal Component Analysis based on prey proportions identified distinct perilithon grazer and benthophagous behaviours. Furthermore, our results were consistent with the available literature on feeding behaviour in these fish. The simplicity of the PCR-based method and its potential generalization to other freshwater organisms may open new perspectives in food web ecology.     

Characterization of human intestinal bifidobacteria using competitive PCR and PCR-TTGE

In this study, a competitive PCR was developed to estimate the quantity of bifidobacteria in human faecal samples using two 16S rRNA gene Bifidobacterium genus-specific primers, Bif164f and Bif662r. A PCR-temporal temperature gradient gel electrophoresis (TTGE) with the same primers also allowed us to describe the Bifidobacterium species present in these faecal samples. The PCR product obtained from the competitor had 467 bp, and was 47 bp shorter than the PCR products obtained from Bifidobacterium strains. The number of bifidobacterial cells was linear from 10 to 10(8) cells per PCR assay. Taking into account the dilutions of the extracted DNA, the linear range was over 8 x 10(5) bifidobacteria g(-1) of faeces. Reproducibility was assessed from 10 independent DNA extractions from the same stool and the coefficient of variation was 0.5%. When the competitive PCR was compared with the culture method, a similar count of seven out of nine Bifidobacterium pure cultures were obtained, or had a difference inferior or equal to 1 log(10). In faecal samples, the enumeration of Bifidobacterium genus in most cases gave higher results with competitive PCR than with culture on selective Columbia-Beerens agar pH 5 (P < 0.05). In conclusion, this competitive PCR allows a rapid, highly specific and reproducible quantification of Bifidobacterium genus in faecal samples. TTGE fragments co-migrating with B. longum CIP64.63 fragment were found in 10 out of 11 faecal samples. Bifidobacterium adolescentis and B. bifidum were detected in five out of 11 subjects. Thus, cPCR and PCR-TTGE can be associated in order to characterize human faecal bifidobacteria.     

New Group-Specific 16S rDNA Primers for Monitoring Foaming Mycolata During Saline Waste-Water Treatment

Newly designed group-specific PCR primers for denaturing gradient gel electrophoresis (DGGE) were used to investigate foaming mycolata from a bioreactor treating an industrial saline waste-water. Genetic profiles on DGGE gels were different with NaCl at 1.65 and 8.24 g l⁻¹, demonstrating that mycolata community was affected by salinity. A semi-nested PCR strategy resulted in more bands in community genetic profiles than direct amplification. DNA sequencing of bands confirmed the efficacy of the novel primers with sequences recovered being most similar to foam producing mycolata. The new group-specific primers/DGGE approach is a new step toward a more complete understanding of functionally important groups of bacteria involved in biological treatment of waste-water. Revisions requested 1 December 2005; Revisions received 19 December 2005     

Molecular analysis of intestinal microbiota of rainbow trout (Oncorhynchus mykiss)

The aim of this study was to evaluate different molecular tools based on the 16S rRNA gene, internal transcribed spacer, and the rpo B gene to examine the bacterial populations present in juvenile rainbow trout intestines. DNA was extracted from both pooled intestinal samples and bacterial strains. Genes were PCR-amplified and analysed using both temporal temperature gradient gel electrophoresis (TTGE) and restriction fragment length polymorphism methods. Because of the high cultivability of the samples, representative bacterial strains were retrieved and we compared the profiles obtained from isolated bacteria with the profile of total bacteria from intestinal contents. Direct analysis based on rpo B-TTGE revealed a simple bacterial composition with two to four bands per sample, while the 16S rRNA gene-TTGE showed multiple bands and comigration for a few species. Sequencing of the 16S rRNA gene- and rpo B-TTGE bands revealed that the intestinal microbiota was dominated by Lactococcus lactis, Citrobacter gillenii, Kluyvera intermedia, Obesumbacterium proteus , and Shewanella marinus . In contrast to 16S rRNA gene-TTGE, rpo B-TTGE profiles derived from bacterial strains produced one band per species. Because the single-copy state of rpo B leads to a single band in TTGE, the rpo B gene is a promising molecular marker for investigating the bacterial community of the rainbow trout intestinal microbiota.     

A previously described serum protein polymorphism in the rat identified as Gc ('vitamin D-binding protein')

The previously published serum protein polymorphisms Gl-1 (Moutier, Toyama & Charrier, 1973) and tf (Bender & Gunther, 1978) are identical and represent genetic variation at the locus of the vitamin D-binding arglobulin, also known as Gc or group-specific component. The identity was established by comparative protein staining, by functional tests with ^;4C-vitamin D₃, by immunological studies with specific anti-Gc sera and by the strain distribution patterns. The Gc polymorphism in the rat may initiate interesting physiological and genetical studies.     

Methanotrophic Communities in the Soils of the Russian Northern Taiga and Subarctic Tundra

The PCR analysis of DNA extracted from soil samples taken in the Russian northern taiga and subarctic tundra showed that the DNA extracts contain genes specific to methanotrophic bacteria, i.e., the mmoX gene encoding the conserved -subunit of the hydroxylase component of soluble methane monooxygenase, the pmoA gene encoding the -subunit of particulate methane monooxygenase, and the mxaFgene encoding the -subunit of methanol dehydrogenase. PCR analysis with group-specific primers also showed that methanotrophic bacteria in the northern taiga and subarctic tundra soils are essentially represented by the type I genera Methylobacter, Methylomonas, Methylosphaera, and Methylomicrobium and that some soil samples contain type II methanotrophs close to members of the genera Methylosinus and Methylocystis. The electron microscopic examination of enrichment cultures obtained from the soil samples confirmed the presence of methanotrophic bacteria in the ecosystems studied and showed that the methanotrophs contain only small amounts of intracytoplasmic membranes.     

Localization of major group-specific polypeptides in the avian RNA tumor viruses

Chloramine T iodination of intact avian myeloblastosis virus revealed, that the four major group-specific polypeptides were labeled unequally, with the largest polypeptide 4 (m.w. 23,000) being preferentially iodinated, when the labeling time was limited. By contrast, in enzymatic iodination of intact virus only major virion glycoprotein and glycolipids were extensively iodinated. These observations suggest that the protein 4 surrounds other group-specific polypeptides and is itself enveloped by the bilayer of glycolipids.     

2B4+ CD8+ T cells play an inhibitory role against constrained HIV epitopes

Cytotoxic T cells play a critical role in the control of HIV and the progression of infected individuals to AIDS. 2B4 (CD244) is a member of the SLAM family of receptors that regulate lymphocyte development and function. The expression of 2B4 on CD8⁺ T cells was shown to increase during AIDS disease progression. However, the functional role of 2B4⁺ CD8⁺ T cells against HIV infection is not known. Here, we have examined the functional role of 2B4⁺ CD8⁺ T cells during and after stimulation with HLA B14 or B27 restricted HIV epitopes. Interestingly, IFN-γ secretion and cytotoxic activity of 2B4⁺ CD8⁺ T cells stimulated with HIV peptides were significantly decreased when compared to influenza peptide stimulated 2B4⁺ CD8⁺ T cells. The expression of the signaling adaptor molecule SAP was downregulated in 2B4⁺ CD8⁺ T cells upon HIV peptide stimulation. These results suggest that 2B4⁺ CD8⁺ T cells play an inhibitory role against constrained HIV epitopes underlying the inability to control the virus during disease progression.