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R J Gast

Publications and source records attributed to R J Gast.

8 recordsLinked to original sources
PubMed2006

DGGE-based detection method for Quahog Parasite Unknown (QPX).

Quahog Parasite Unknown (QPX) is a significant cause of hard clam Mercenaria mercenaria mortality along the northeast coast of the United States. It infects both wild and cultured clams, often annually in plots that are heavily farmed. Subclinically infected clams can be identified by histological examination of the mantle tissue, but there is currently no method available to monitor the presence of QPX in the environment. Here, we report on a polymerase chain reaction (PCR)-based method that will facilitate the detection of QPX in natural samples and seed clams. With our method, between 10 and 100 QPX cells can be detected in 1 l of water, 1 g of sediment and 100 mg of clam tissue. Denaturing gradient gel electrophoresis (DGGE) is used to establish whether the PCR products are the same as those in the control QPX culture. We used the method to screen 100 seed clams of 15 mm, and found that 10 to 12% of the clams were positive for the presence of the QPX organism. This method represents a reliable and sensitive procedure for screening both environmental samples and potentially contaminated small clams.

Animals↗
PubMed1996

Molecular phylogeny of symbiotic dinoflagellates from planktonic foraminifera and radiolaria.

Recent analyses of the small subunit ribosomal DNA (srDNA) from dinoflagellate symbionts of cnidaria have confirmed historical descriptions of a diverse but well-defined clade, Symbiodinium, as well as several other independent symbiont lineages (Rowan 1991; Rowan and Powers 1992; Sadler et al. 1992; McNally et al 1994). Dinoflagellates also occur as intracellular symbionts in a number of pelagic protistan taxa, but the srDNA of these symbionts has not been examined. We analyzed the srDNA sequences of the symbiotic dinoflagellates from four planktonic foraminiferal species and six radiolarian species. The symbionts from these sarcodines formed two distinct lineages within the dinoflagellates. Within each lineage, symbionts obtained from different host species showed few, if any, srDNA sequence differences. The planktonic foraminiferal symbionts were most closely related to Gymnodinium simplex and the Symbiodinium clade, whereas the radiolarian symbionts were most closely related to the dinoflagellate symbiont from the oceanic chondrophore, Velella velella. Therefore, although the dinoflagellate symbionts of foraminifera appear to be a sister taxon of the symbionts from benthic foraminifera and invertebrates, the symbionts of radiolaria are distinct and arose from an independent lineage of dinoflagellate symbionts that shares common ancestry with the symbiont of at least one pelagic metazoan. The lack of srDNA variability within the sarcodine symbiont lineages suggests that coevolution of host and symbiont has not occurred.

Animals↗
PubMed1994

Discovery of group I introns in the nuclear small subunit ribosomal RNA genes of Acanthamoeba.

The discovery of group I introns in small subunit nuclear rDNA (nsrDNA) is becoming more common as the effort to generate phylogenies based upon nsrDNA sequences grows. In this paper we describe the discovery of the first two group I introns in the nsrDNA from the genus Acanthamoeba. The introns are in different locations in the genes, and have no significant primary sequence similarity to each other. They are identified as group I introns by the conserved P, Q, R and S sequences (1), and the ability to fit the sequences to a consensus secondary structure model for the group I introns (1, 2). Both introns are absent from the mature srRNA. A BLAST search (3) of nucleic acid sequences present in GenBank and EMBL revealed that the A. griffini intron was most similar to the nsrDNA group I intron of the green alga Dunaliella parva. A similar search found that the A. lenticulata intron was not similar to any of the other reported group I introns.

Acanthamoeba↗
PubMed1994

Sequence variations in small-subunit ribosomal RNAs of Hartmannella vermiformis and their phylogenetic implications.

Evidence of associations between free-living amoebas and human disease has been increasing in recent years. Knowledge about phylogenetic relationships that may be important for the understanding of pathogenicity in the genera involved is very limited at present. Consequently, we have begun to study these relationships and report here on the phylogeny of Hartmannella vermiformis, a free-living amoeba that can harbor the etiologic agent of Legionnaires' disease. Our analysis is based on studies of small-subunit ribosomal RNA genes (srDNA). Nucleotide sequences were determined for nuclear srDNA from three strains of H. vermiformis isolated from the United Kingdom, Germany, and the United States. These sequences then were compared with a sequence previously obtained for a North American isolate by J. H. Gunderson and M. L. Sogin. The four genes are 1,840 bp long, with an average GC content of 49.6%. Sequence differences among the strains range are 0.38%-0.76%. Variation occurs at 19 positions and includes 2 single-base indels plus 14 monotypic and 3 ditypic single-base substitutions. Variation is limited to eight helix/loop structures according to a current model for srRNA secondary structure. Parsimony, distance, and bootstrap analyses used to examine phylogenetic relationships between the srDNA sequences of H. vermiformis and other eukaryotes indicated that Hartmannella sequences were most closely related to those of Acanthamoeba and the alga Cryptomonas. All ditypic sites were consistent with a separation between European and North American strains of Hartmannella, but results of other tests of this relationship were statistically inconclusive.

Acanthamoeba↗
PubMedDate not supplied

Subgenus systematics of Acanthamoeba: four nuclear 18S rDNA sequence types.

Classification of Acanthamoeba at the subgenus level has been problematic, but increasing reports of Acanthamoeba as an opportunistic human pathogen have generated an interest in finding a more consistent basis for classification. Thus, we are developing a classification scheme based on RNA gene sequences. This first report is based on analysis of complete sequences of nuclear small ribosomal subunit RNA genes (Rns) from 18 strains. Sequence variation was localized in 12 highly variable regions. Four distinct sequence types were identified based on parsimony and distance analyses. Three were obtained from single strains: Type T1 from Acanthamoeba castellanii V006, T2 from Acanthamoeba palestinensis Reich, and T3 from Acanthamoeba griffini S-7. T4, the fourth sequence type, included 15 isolates classified as A. castellanii, Acanthamoeba polyphaga, Acanthamoeba rhysodes or Acanthamoeba sp., and included all 10 Acanthamoeba keratitis isolates. Interstrain sequence differences within T4 were 0%-4.3%, whereas differences among sequence types were 6%-12%. Branching orders obtained by parsimony and distance analyses were inconsistent with the current classification of T4 strains and provided further evidence of a need to reevaluate criteria for classification in this genus. Based on this report and others in preparation, we propose that Rns sequence types provide the consistent quantititive basis for classification that is needed.

Acanthamoeba↗
PubMedDate not supplied

The evolutionary history of the genus Acanthamoeba and the identification of eight new 18S rRNA gene sequence types.

The 18S rRNA gene (Rns) phylogeny of Acanthamoeba is being investigated as a basis for improvements in the nomenclature and taxonomy of the genus. We previously analyzed Rns sequences from 18 isolates from morphological groups 2 and 3 and found that they fell into four distinct evolutionary lineages we called sequence types T1-T4. Here, we analyzed sequences from 53 isolates representing 16 species and including 35 new strains. Eight additional lineages (sequence types T5-T12) were identified. Four of the 12 sequence types included strains from more than one nominal species. Thus, sequence types could be equated with species in some cases or with complexes of closely related species in others. The largest complex, sequence type T4, which contained six closely related nominal species, included 24 of 25 keratitis isolates. Rns sequence variation was insufficient for full phylogenetic resolution of branching orders within this complex, but the mixing of species observed at terminal nodes confirmed that traditional classification of isolates has been inconsistent. One solution to this problem would be to equate sequence types and single species. Alternatively, additional molecular information will be required to reliably differentiate species within the complexes. Three sequence types of morphological group 1 species represented the earliest divergence in the history of the genus and, based on their genetic distinctiveness, are candidates for reclassification as one or more novel genera.

Acanthamoeba↗
PubMedDate not supplied

Development of an Acanthamoeba-specific reverse dot-blot and the discovery of a new ribotype.

Acanthamoeba is a genus of free-living amoebae, of which some species have been found to cause opportunistic infections in humans. The identification of these amoebae in natural and disease samples is based primarily upon morphological features. While these features are more than adequate for identification to the genus level, they are not useful for species-level identification. This not only leads to difficulty in the diagnosis of infections, but it makes an accurate assessment of the natural distribution of acanthamoebae very difficult to achieve. To improve this situation, a detection method was developed that utilizes both selective polymerase chain reaction amplification and the reverse dot-blot. Oligonucleotides were designed to be specific for the described ribosomal groups (or ribotypes) of Acanthamoeba, as well as one specific for the genus itself. When this method was used to analyze a series of Acanthamoeba cultures from Pakistan, a new ribotype was identified in addition to the detection of the ubiquitously distributed T4 type.

Acanthamoeba↗