Multiple polymorphic sites at the ITS and ATAN loci in cultured isolates of Perkinsus marinus
Gwynne D Brown1, Karen L Hudson, Kimberly S Reece
1Virginia Institute of Marine Science, College of William and Mary, P.O. Box 1346, Gloucester Point, Virginia 23062, USA.
Abstract:
Sequence analysis of genomic DNA from the protozoan parasite Perkinsus marinus at two loci revealed genetic polymorphisms within and among different cultured isolates. Genomic DNA from 12 Perkinsus marinus isolates was amplified at the internal transcribed spacer region and at an anonymous locus previously identified to contain polymorphisms by restriction fragment length polymorphism analysis. Fourteen polymorphic nucleotide positions were identified at the internal transcribed spacer region; eight in internal transcribed spacer 1 and six in internal transcribed spacer 2. Thirteen polymorphic nucleotide sites were identified within the anonymous locus. In some instances, more than three different sequences were observed at both the internal transcribed spacer region and at the anonymous locus from a single clonal isolate, suggesting the possibility of recombination in cultured cells and/or strand jumping during the polymerase chain reaction. Intra-isolate sequence variation (3.46% for the anonymous locus and 3.08% for internal transcribed spacer 1) was in several cases as high as inter-isolate sequence variation, even in one isolate where recombination was not evident. High intra- and inter-isolate variation detected at both loci demonstrates the importance of determining the genetic variation of each locus prior to development of sequence-based molecular diagnostics.
Insights
Genetic analysis of Perkinsus marinus revealed significant DNA variation within and between isolates. This high intra-isolate sequence variation highlights the need for careful genetic assessment before developing molecular diagnostics.
Area of Science:
- Genomics
- Parasitology
- Molecular Biology
Background:
- Perkinsus marinus is a protozoan parasite impacting marine bivalves.
- Understanding genetic diversity is crucial for effective parasite management and diagnostics.
- Previous studies identified genetic polymorphisms using restriction fragment length polymorphism analysis.
Purpose of the Study:
- To analyze genetic polymorphisms in Perkinsus marinus at the internal transcribed spacer (ITS) region and an anonymous locus.
- To assess the extent of intra-isolate and inter-isolate genetic variation.
- To evaluate the implications of observed genetic variation for developing sequence-based molecular diagnostics.
Main Methods:
- Genomic DNA extraction from 12 cultured Perkinsus marinus isolates.
- DNA amplification of the internal transcribed spacer (ITS) region and an anonymous locus using polymerase chain reaction (PCR).
- DNA sequence analysis to identify polymorphic nucleotide positions and assess sequence variation.
Main Results:
- Fourteen polymorphic nucleotide positions were identified in the ITS region (8 in ITS 1, 6 in ITS 2).
- Thirteen polymorphic nucleotide sites were identified within the anonymous locus.
- High intra-isolate sequence variation (up to 3.46%) was observed, comparable to inter-isolate variation, suggesting potential recombination or PCR artifacts.
Conclusions:
- Significant genetic polymorphisms exist within and among Perkinsus marinus isolates.
- High intra-isolate variation necessitates thorough genetic characterization of each locus.
- Sequence-based molecular diagnostics for Perkinsus marinus require careful consideration of observed genetic diversity.
Related Concept Videos
01:19Allosteric Proteins-ATCase
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...
00:49Ligand Binding and Linkage
01:29Modern Molecular Taxonomy