Identification of Two Large-Subunit rRNAs by RT-PCR from A Strain Of the Marine Dinoflagellate Alexandrium tamerense




Rami Alsaber

Introduction

Many marine dinoflagellate species that are part of the genus Alexandrium (= Protogonyaulax) represent a significant portion of the bloom-forming common agents of toxic red tides. Due to the damaging effects of these toxins, much work has been done on the ecology and physiology of toxin biosynthesis in certain toxic strains, and the monitoring and prediction of outbreaks of blooms caused by these organisms. A determinant in studying bloom dynamics is the calculation of the in situ rates of growth of these organisms under varying conditions.

Only in the past few years have we began to see more research focusing on gene regulation and in other mechanisms in dinoflagellates in an attempt to develop new methods for measuring and predicting cell growth rates. The goal of this study is to isolate the LsurRNA itself and detect any variation within growth-synchronized non-toxic strain of the species Alexandrium tamarense. This task will involve direct isolation of total RNA from healthy cultures, amplification of selected RNA targets by RT-PCR, and the sequencing of the RT-PCR products to analyze and distinguish any variation.

Figures


Figure 1-RNA extraction detection. Lane L is a 1Kb ladder. Lanes I and II are of a non-toxic strain of A. Tamerense RNA collected 2 and 12 hr after blocking period respectively.


Figure 2-RT-PCR products for LsurRNA of A. Tamarense . Lane L is a 100bp ladder. Lanes I and II are of a non-toxic strain of A. Tamerense RNA collected 2 and 12 hr respectively. Lane C is a control reaction lacking sample RNA. Lane I and II share a ~550bp band corresponding to the LsurRNA. In Lane II, a smaller band ~450bp was detected in these cells.


Figure 3-Gel-comparison of gel-extracted DNA bands from RT-PCR. Lanes 1, 2 and 3 contain DNA samples with known concentrations of 10, 20, and 50 ug/ml respectively. Lane 4 contains 2ul of 30ul elution of the extracted ~450bp band from RT-PCR gel. Lanes 5 contains 2ul of 30ul elution of the ~550bp band found in RT-PCR gel lanes II (Fig. 2). Lane L is a 100bp ladder.


Figure 4-Partial sequence analysis of the two LsurRNA bands isolated from RT-PCR reaction compared to published A. tamarense LsurRNA gene. LsurRNA1 represent the large-band sequence. LsurRNA2 represents the small-band sequence.


Batch cultures of the non-toxic strain of A. tamarense were used in this study. Cultures were synchronized by a dark-induced block/release method. Samples (3x106 cells) for RNA analysis were taken 2 and 12 hours after the blocking period. RNA was extracted from all samples and its presence was confirmed by gel-electrophoresis (Fig. 1). Successful RNA extraction was observed in both strains early and in late G1 phase post the dark blocking. Samples from all RNA extractions underwent PCR amplification to identify any negative DNA contamination and no detectable bands were found. RT-PCR for LsurRNA was carried on for 45 cycles of amplification. A smaller LsurRNA band was detected in cells that were extracted 12 hr after the dark blocking period (Fig. 2). DNA bands from the RT-PCR gel were removed and purified and the concentration of their elution was determined by gel-comparison with DNA samples of known concentrations (Fig. 3). Sequencing was performed on the bands corresponding to LsurRNA and the small additional band found in one of the samples. Sequence analysis and comparison to published LsurRNA gene showed almost identical sequences between LsurRNA bands and great homology with the detected smaller band (Fig. 4). Further confirmation of the identity of the LsurRNA bands was achieved by ligation and cloning into a Promega™ PGEM®-T Easy vector. The cloned insert was amplified and sequenced, and it confirmed the identity of the band to LsurRNA.
In this study we observe the detection of an expressed variant of LsurRNA that is present at a later cell-cycle stage/growth phase in synchronized cultures of non-toxic Alexandrium tamarense. Dinoflagellate chromosomes are known for having multiple gene copies for many constitutive and differentially expressed genes. LsurRNA gene sequence data from previous studies suggested that the Alexandrium species complex was a heterogeneous group. However, any morphological similarities were not confirmed by sequence similarities. This is evident in the slight 3- or 4-bp difference seen between the LsurRNA sequenced in this study and the published LsurRNA sequences as well as slight differences between the smaller band and the large LsurRNA detected. Based on this variation, developing detection and quantitative techniques that are based on presence of amount-specific oligonucleotide probes for LsurRNA could be challenging. Little data on gene regulation in marine dinoflagellates exist. Earlier studies show that dinoflagellates are capable of packaging portion of their genome which intern allows for horizontal gene transfer among members of the genus. This could possibly explains the close genetic similarities among different Alexandrium species within a specific region. More recent studies have shown that transcriptional and pretranslationtional regulation does occur in dinoflagellates. In conclusion, transcriptional regulation in dinoflagellate can no longer be overlooked. Genetic content within each strain can vary and their expression can differ throughout the various stages of the cell cycle. Here we show that the use of RT-PCR to look for transcription products can be used to observe the various cell-cycle control mechanisms and even the regulation of toxin biosynthesis.

Full Paper

Acknowledgments

I would like to thank Dr. Berish Rubin of the biology department of Fordham University for his monitoring and guidance throughout the course of this study. I would also like to thank Mr. Jinsong Qiu and Mr. Brian Fox for their help and assistance with the various molecular methods and techniques employed throughout this study.


This document was last modified 01/31/2006.
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