RESUMO
A comparison of the small subunit rRNA sequences of a Chesapeake Bay strain of the dinoflagellate Akashiwo sanguinea and the dinoflagellate Amoebophrya sp. parasitizing it revealed several potential target sites that could be used to detect the parasite through in situ hybridization. The fluorescence of probed cells under various conditions of hybridization was measured by using a spot meter on a Nikon UFX-II camera attachment so that the effect of various hybridization parameters on probe binding could be determined. Probes directed against both the junction between helices 8 and 11 and helix 46 could detect the parasite, although the helix 8/11 probe produced a stronger signal under the conditions tested. The fluorescence of the probed cells increased with increasing hybridization time up to approximately twelve hours. The background fluorescence was lower at the wavelengths used to detect Texas Red than at those used to detect fluorescein, so probed cells were more distinct when Texas Red was used as the label. Cells stored in cold paraformaldehyde for a year still bound the probes. Young stages of the parasite could be seen more readily after in situ hybridization than after protargol impregnation.
Assuntos
Sondas de DNA , Dinoflagellida/genética , Dinoflagellida/parasitologia , Hibridização in Situ Fluorescente , Água do Mar/parasitologia , Animais , Sequência de Bases , Dinoflagellida/química , Interações Hospedeiro-Parasita , Maryland , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA de Protozoário/química , RNA de Protozoário/genética , RNA Ribossômico/química , RNA Ribossômico/genética , XantenosRESUMO
The small-subunit rRNA sequence of a species of Amoebophrya infecting Gymnodinium sanguineum in Chesapeake Bay was obtained and compared to the small subunit rRNA sequences of other protists. Phylogenetic trees constructed with the new sequence place Amoebophrya between the remaining dinoflagellates and other protists.
Assuntos
Dinoflagellida/genética , Dinoflagellida/parasitologia , Filogenia , RNA Ribossômico/genética , Animais , Dinoflagellida/classificação , Genes de RNAr , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
In situ hybridization using fluorescent oligonucleotide probes complementary to unique regions of 16S rRNA molecules provides a way of identifying the food vacuole contents of bactivorous protists. Laboratory experiments with Tetrahymena showed rRNAs in food vacuoles are degraded slowly enough to permit their use as hybridization targets for such probes. A probe specific for a hypervariable region of the small subunit rRNA of an unnamed proteobacterium abundant in a local lake was then synthesized. It was used to probe the food vacuoles of the ciliates present in fixed water samples collected from the same lake. The vacuoles of several filter-feeding ciliates bound the probe, indicating that such probes can be used to identify the food vacuole contents of ciliates collected from natural samples.
