Real-time quantitative broad-range PCR assay for detection of the 16S rRNA gene followed by sequencing for species identification

Here we determined the analytical sensitivities of broad-range real-time PCR-based assays employing one of three different genomic DNA extraction protocols in combination with one of three different primer pairs targeting the 16S rRNA gene to detect a panel of 22 bacterial species. DNA extraction protocol III, using lysozyme, lysostaphin, and proteinase K, followed by PCR with the primer pair Bak11W/Bak2, giving amplicons of 796 bp in length, showed the best overall sensitivity, detecting DNA of 82% of the strains investigated at concentrations of < or =10(2) CFU in water per reaction. DNA extraction protocols I and II, using less enzyme treatment, combined with other primer pairs giving shorter amplicons of 466 bp and 342 or 346 bp, respectively, were slightly more sensitive for the detection of gram-negative but less sensitive for the detection of gram-positive bacteria. The obstacle of detecting background DNA in blood samples spiked with bacteria was circumvented by introducing a broad-range hybridization probe, and this preserved the minimal detection limits observed in samples devoid of blood. Finally, sequencing of the amplicons generated using the primer pair Bak11W/Bak2 allowed species identification of the detected bacterial DNA. Thus, broad-spectrum PCR targeting the 16S rRNA gene in the quantitative real-time format can achieve an analytical sensitivity of 1 to 10 CFU per reaction in water, avoid detection of background DNA with the introduction of a broad-range probe, and generate amplicons that allow species identification of the detected bacterial DNA by sequencing. These prerequisites are important for its application to blood-containing patient samples.

Fluorescent in situ hybridization mapping of the epidermal growth factor receptor gene in donkey

The physical localization of the epidermal growth factor receptor (EGFR) gene was performed on donkey chromosomes. Bacterial artificial chromosome DNA containing the equine EGFR gene was used to map this gene by fluorescent in situ hybridization on donkey metaphase chromosomes. The gene was mapped on donkey 1q21.1 region.

Very short telomere length by flow fluorescence in situ hybridization identifies patients with dyskeratosis congenita

Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome in which the known susceptibility genes (DKC1, TERC, and TERT) belong to the telomere maintenance pathway; patients with DC have very short telomeres. We used multicolor flow fluorescence in situ hybridization analysis of median telomere length in total blood leukocytes, granulocytes, lymphocytes, and several lymphocyte subsets to confirm the diagnosis of DC, distinguish patients with DC from unaffected family members, identify clinically silent DC carriers, and discriminate between patients with DC and those with other bone marrow failure disorders. We defined "very short" telomeres as below the first percentile measured among 400 healthy control subjects over the entire age range. Diagnostic sensitivity and specificity of very short telomeres for DC were more than 90% for total lymphocytes, CD45RA+/CD20- naive T cells, and CD20+ B cells. Granulocyte and total leukocyte assays were not specific; CD45RA- memory T cells and CD57+ NK/NKT were not sensitive. We observed very short telomeres in a clinically normal family member who subsequently developed DC. We propose adding leukocyte subset flow fluorescence in situ hybridization telomere length measurement to the evaluation of patients and families suspected to have DC, because the correct diagnosis will substantially affect patient management.

Telomere length in human natural killer cell subsets

Natural killer (NK) cells are cytotoxic cells that play a critical role in the innate immune response against infections and tumors. In the elderly, the cytotoxic function of NK cells is often compromised. Telomeres progressively shorten with each cell division and with age in most somatic cells eventually leading to chromosomal instability and cellular senescence. We studied the telomere length in NK cell subsets isolated from peripheral blood using "flow FISH," a method in which the hybridization of telomere probe in cells of interest is measured relative to internal controls in the same tube. We found that the average telomere length in human NK cells decreased with age as was previously found for human T lymphocytes. Separation of adult NK cells based on CD56 and CD16 expression revealed that the telomere length was significantly shorter in CD56(dim)CD16(+) (mature) NK cells compared to CD56(bright)CD16(-) (immature) NK cells from the same donor. Furthermore, sorting of NK cells based on expression of activation markers, such as NKG2D and LFA-1, revealed that NK cells expressing these markers have significantly shorter telomeres. Telomere fluorescence was very heterogeneous in NK cells expressing CD94, killer inhibitory receptor (KIR), NKG2A, or CD161. Our observations indicate that telomeric DNA in NK cells is lost with cell division and with age similar to what has been observed for most other hematopoietic cells. Telomere attrition in NK cells is a plausible cause for diminished NK cell function in the elderly.

Hybridization and Introgression Between Native and Alien Species

Human activities, such intentional and unintentional transplantations, and habitat alterations including the establishment of migration corridors, generate increasing opportunities for formerly allopatric taxa to meet and to hybridize. There is indeed increasing evidence that these introduced plant and animal taxa (including crop plants and domesticated animal taxa) frequently hybridize with native relatives and with other introduced taxa, leading to a growing concern that these hybridizations may compromise the genetic integrity of native taxa to the point of causing extinctions (Abbott 1992; Rhymer and Simberloff 1996; Levin et al. 1996; Ellstrand and Schierenbeck 2000; Vilà et al. 2000). A decade ago, Rhymer and Simberloff (1996) stated in their review on this topic that the known cases are probably just the tip of the iceberg.Using the search term ‘hybridization and introgression’, the Web of Science database yields a total of 1,178 research articles, of which 935 (or 80 %) have been published after 1995 (Fig. 16.1). Indeed, the evidence for natural and man-induced hybridization and introgression appears to have increased exponentially these last few years.

Speciation reversal and biodiversity dynamics with hybridization in changing environments

A considerable fraction of the world's biodiversity is of recent evolutionary origin and has evolved as a by-product of, and is maintained by, divergent adaptation in heterogeneous environments. Conservationists have paid attention to genetic homogenization caused by human-induced translocations (e.g. biological invasions and stocking), and to the importance of environmental heterogeneity for the ecological coexistence of species. However, far less attention has been paid to the consequences of loss of environmental heterogeneity to the genetic coexistence of sympatric species. Our review of empirical observations and our theoretical considerations on the causes and consequences of interspecific hybridization suggest that a loss of environmental heterogeneity causes a loss of biodiversity through increased genetic admixture, effectively reversing speciation. Loss of heterogeneity relaxes divergent selection and removes ecological barriers to gene flow between divergently adapted species, promoting interspecific introgressive hybridization. Since heterogeneity of natural environments is rapidly deteriorating in most biomes, the evolutionary ecology of speciation reversal ought to be fully integrated into conservation biology.