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Isolation and characterization of serine protease gene(s) from Perkinsus marinus
Gwynne D Brown1, Kimberly S Reece
1Virginia Institute of Marine Science, PO Box 1346, Gloucester Point, Virginia 23062, USA.
Abstract:
This study reports the first serine protease gene(s) isolated from Perkinsus marinus. Using universal primers, a 518 bp subtilisin-like serine protease gene fragment was amplified from P. marinus genomic DNA and used as a probe to screen a lambda-phage P. marinus genomic library; 2 different lambda-phage clones hybridized to the digoxigenin(DIG)-labeled subtilisin-like gene fragment. Following subcloning and sequencing of the larger DNA fragment, a 1254 bp open reading frame was identified and later confirmed, by 5' and 3' random amplification of cDNA ends (RACE) and northern blot analysis, to contain the entire coding-region sequence. Sequence analysis of the 3' RACE results from 2 isolate cultures, VA-2 (P-1) and LA 10-1, revealed multiple polymorphic sites within and among isolates. We identified 2 different types of cDNA clones with 95.53% nucleotide sequence similarity, suggesting the possibility of 2 closely related genes within the P. marinus genome. Southern blot analysis of genomic DNA from 12 genetically distinct P. marinus isolate cultures revealed 2 different banding patterns among isolates.
Insights
Researchers identified the first serine protease genes in Perkinsus marinus. Analysis revealed two closely related genes, suggesting genetic diversity within this important marine parasite.
Area of Science:
- Molecular Biology
- Parasitology
- Marine Biology
Background:
- Perkinsus marinus is a significant pathogen affecting oysters.
- Understanding the genetic makeup of P. marinus is crucial for disease management.
- Serine proteases are important enzymes involved in various biological processes.
Purpose of the Study:
- To isolate and characterize serine protease gene(s) from Perkinsus marinus.
- To investigate the genetic diversity of serine protease genes within P. marinus isolates.
Main Methods:
- Amplification of a serine protease gene fragment using universal primers.
- Screening of a P. marinus genomic library with a labeled gene probe.
- Sequencing, 5' and 3' RACE, Northern blot, and Southern blot analyses.
Main Results:
- A 1254 bp open reading frame encoding a subtilisin-like serine protease was identified.
- Sequence analysis revealed two distinct cDNA clones with 95.53% nucleotide similarity, indicating two related genes.
- Southern blot analysis showed two different banding patterns among 12 P. marinus isolates, supporting genetic variation.
Conclusions:
- This study reports the first isolation and characterization of serine protease gene(s) from P. marinus.
- The findings suggest the presence of at least two closely related serine protease genes in the P. marinus genome.
- Genetic polymorphism in serine protease genes may contribute to the diversity observed among P. marinus isolates.
