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Comparative study of algal DNA in Cymbella sp by restriction fragment length polymorphism.

Introduction

Algae alter their gene expression in response to environmental changes or external signals to switch between coherent genetic programs in order to produce a phenotypic state, among many available that best copes with the new environment. It is increasingly becoming clear that such genetic programs represent attractor states: discrete stable states of gene expression patterns generated by the dynamics of the regulatory interactions between the genes. Environmental conditions are much larger than that of cellular response programs, there is not a program for each condition, and cells need to choose the optimal program for a given condition. Thus, many environmental conditions map onto the same cellular response. The ability to respond rapidly to fluctuations in temperature, nutrients, and other environmental changes is important for competitive fitness and cell survival. Understanding the response of cells to environmental changes is of interest because it can provide clues to the molecular apparatuses that enable cells to adapt to new environments and the molecular mechanisms that have evolved to regulate the remodeling of gene expression that occurs in new environments. Significant clues to the mechanisms involved in adaptation to new environments have come from studies of the genes that are expressed in response to specific stresses. For example, cells exposed to elevated temperatures induce transcription of genes encoding heat shock proteins (Craig, 1992) 4. Cells must coordinate adjustments in genome expression to accommodate changes in their environment. Despite our lack of knowledge about the complete set of genes involved in these changes, investigators have identified transcriptional activators and repressors that likely contribute to coordinate remodeling of genome expression.

Cells living in variable environments must adapt to sudden environmental changes that can stress the cellular system. Most natural environments are inherently variable over space and time, and as such environmental stresses can occur across gradients, in combinations, and in rapid succession. The ability to survive successive stress treatments and to prepare for severe stress after early signs of a problem presents a significant selective advantage for creatures living in natural environments.

The dissection of the sources of variation which account for the total phenotypic variability in an organism is difficult to accomplish with field populations. It is possible, however, to minimize variability by cultivation of live material under controlled laboratory conditions. Experimentation with clones, for example, makes possible the distinction between exclusively genetic and genotype-by-environment variability in a genetically homogeneous background. Other methodologies include the use of mutants with promising results in plants and cyanobacteria (Clack et al. 1994, Kehoe and Grossman 1996) [3]. Environmental effects upon the genetic machinery are brought about by various intracellular and extracellular factors operating throughout the life history of organisms. These factors elicit genetic responses that ultimately manifest themselves through a modification (physiological and/or morphological) of the phenotype. In other cases, as takes place with the green alga Scenedesmus, morphological and physiological changes are observed from parents to offspring (Morales and Trainor, 1999) [15]. Genetic responses to environmental changes are coordinated through an epigenetic system capable of regulating the functioning of its parts (genes). The effect of such functioning is the "calibration" of an organism to the new surrounding conditions. Hence, many phenotypic characteristics are the end result of gene activity, with the amount and direction modulated by environmental variability. Genetic sources of phenotypic variation include all those processes altering the structure and composition of the genetic material of an individual (mutations at the gene and chromosome levels, gene duplication, gene flow, and genetic drift). Such genetic sources (excluding mutations) especially apply to sexually reproducing populations. Phenotypic changes due to environmental effects do not imply alterations in genetic structure and composition (mutations), but rather signal that different portions of the genetic code are expressed. The effects of this turning on and off of genes may, in some ways, be comparable to the appearance of novel traits by mutation. (Pigliucci, M.1996) [16]

Materials and Methods

The alga upon which work has been carried out was isolated in Department of Biotechnology, School of life sciences, Vels University, Chennai. Media and glassware were sterilized and autoclave at 120[degrees]C for 15 minutes. All chemicals were used as analytical grade. All four cultures were made anexic to avoid cross contamination.

Sample collection

Water samples are collected from two different environments is industrial water near textile dye effluent from Walajapet, Vellore district and Temple pond water of Vedagirieswarer Temple, Walajapet, and Vellore district. About one litre of water was collected from the depth of 10cm from the surface of water. The water samples were mixed thoroughly and screened within 48 hours of collection.

Isolation of Algae

1ml of water was taken with 10 ml of sterile F/2 medium. From this serial dilution were made. All dilution were streaked in 2% agar plates and incubated at 21[degrees]C in a light chamber with 2k lux. A total six different algae sp are seen in Temple pond water and only three algae sp are seen in polluted water. Cymbella cistula and Cymbella ventricosa both were common in industrial and Temple pond water.

DNA isolation

Isolated Cymbella cistula and Cymbella ventricosa was grown on F/2 medium and incubated at 21[degrees]C for two weeks. The algal biomass was separated by centrifugation at 10000 rpm for 5minutes and pellet was dried under vacuum and used for DNA extraction. 0.1 g of biomass is extracted with liquid nitrogen. Extracted solution was mixed with 8ml CTAB buffer and 2ml of 10% SDS incubated at 60[degrees]C for 30 minutes. Add equal volume of phenol chloroform(1:1) and centrifuge at 10000 rpm for 5 minutes. Add equal volume of isoamyl alcohol to the supernatant and centrifuge at 12000 rpm for 10 minutes. Add one ml of 70% ethanol to pellet and centrifuge at 12000 rpm for 10 minutes. Dissolve the pellet with Tris buffer. The presence of DNA could confirm by Agarose gel electrophoresis. The extracted DNA was run on 1.2% Agarose gel. The Agarose gel electrophoresis was performed for one hour at 50 volts. The gel viewed under the Trans illuminator (Punekar, et al., 2004) [17]

Estimation of DNA purity and quatification

The DNA isolated from algae cells are usually contaminated with protein, RNA, and salts used during the isolation process. The purity of DNA may be estimated by utilizing the property of the heterocyclic rings of the nucleotides of absorbing light strongly in the UV range. DNA absorbs maximum light energy at about 260 nm. An optical density of 1.0 corresponds to approximately 50 mg/mL of double stranded DNA. The ratio of absorbance viz. A260/A2SO and A2SO/ A260 provides an estimation regarding the purity of DNA. To find out the purity of DNA, make the appropriate dilution with TE buffer, and measure the absorbance at 260 nm and 280 nm. Do not use glass or plastic cuvettes, as lights in the UV range do not pass through these.

Restriction Fragments Length Polymorphism (RFLP) Analysis.

The DNA extracted from two different algae was restricted with Hind3. To the reaction mixture add 11.8 [micro]l of nuclease free water, 2 [micro]l of 10x assay buffer, 0.2C of 100x BSA, 5 [micro]l of template and 1 [micro]l of restriction enzyme. The mixture was incubated at 37[degrees]C for one hour then 65 [degrees]C for 10 minutes. 10-15 [micro]l of digested DNA along with dye were loaded on the gel. Electrophoresis was performed for one hour at 50[degrees]C and viewed under the Trans illuminator. As a marker 1Mb ladder was used to find out the molecular weight of restricted DNA.

Result

Two water samples were collected from polluted water (Textile dye industry) and Temple pond water in Vellore district. From these samples the algae were isolated; of these Cymbella cistula and Cymbella ventricosa both were common in industrial and Temple pond water. Algae were cultured in F/2 medium and pure cultures were maintained as slants.

Isolation of DNA

From the pure cultures, the DNA of both species were isolated and confirmed by Agarose gel electrophoresis. The first four wells loaded with DNA extracted from four different Cymbella sp isolated from two different environments. The fifth well loaded with standard E.coli DNA used as control. The DNA bands were clear and matched with E.coli DNA. The result indicated that all the isolates of four species from two different water samples found to contain DNA with similar molecular weight. Nuclear DNA of Cymbella sp was represented in Fig: 1.

[FIGURE 1 OMITTED]

Estimation of DNA

Extracted DNA samples were estimated from all algal cultures were checked its purity and quantification by using spectrophotometer. About 800-900 ng of DNA is obtained from 1 g of pellet from Cymbella cistula and 300-400 ng from Cymbella ventricosa. The ratio of absorbance at 260 to that at 280 nm (A260/280) of the DNA ranged from 1.8-1.9 for Cymbella cistula and 1.7-1.8 for Cymbella ventricosa.

Restriction Fragments Length Polymorphism (RFLP)

The DNA extracted from two different algae was restricted with enzyme Hind3. The results shows that Cymbella cistula isolated from polluted water (Textile dye industry) and Temple pond water showed similar band pattern. Two similar bands were seen about 500kb and 700 kb respectively. Whereas in the case of Cymbella ventricosa, three different bands are seen about 100kb, 200kb and 700kb in polluted water (Textile dye industry), and three different bands of Cymbella ventricosa in Temple pond water are seen about 100kb, 300kb and 600kb respectively. E.coli DNA shows two bands about 600kb and 800kb with Hind 3 digestion. Purity of the DNA ranged from 1.8-1.9 for Cymbella cistula and 1.7-1.8 for Cymbella ventricosa, both the species having same purity but the quantity of DNA were slightly modified. RFLP analysis of Cymbella sp by HIND3 digestion was represented in Fig: 2.

[FIGURE 2 OMITTED]

Discussion

Genome-wide expression analysis was used to explore how gene expression is remodeled in response to changes in extracellular environment, including changes in temperature, oxidation, nutrients, pH, and osmolarity. We found that approximately two-thirds of the genome is involved in the response to environmental changes. The inclusion of a large fraction of genes in environmental responses reveals the importance of expression remodeling in adapting to environmental changes and implicates a substantial set of genes with previously uncharacterized cellular roles in these responses. We examined Diatom Cymbella cistula and Cymbella ventricosa isolated from two different environments. From the pure cultures the total DNA was isolated. The Diatom chromosomal DNA was separated by caesium chloride in TE buffer column and Diatom DNA were separated into two bands based on the gradients. The lower band is greatly enriched in nuclear DNA, and the upper band is greatly enriched in chloroplast DNA. Nuclear DNA was electrophoresed on 1.2% gel. All bands of four samples were cleared and having same molecular weight. Each genomic DNA was having one or few sites for restriction enzymes. In this study the HIND3 enzyme was used, by the restriction pattern the Cymbella cistula and Cymbella ventricosa having two sites for HIND3. The Cymbella cistula shows similar band pattern on both the environments shows there is no changes in molecular pattern, whereas the Cymbella ventricosa shows three different band in polluted water and three different band in pond water shows that the molecular pattern of same species in two different environments is become changed. Due the chemical usage of industrial development the genomic program is changed in an organism. This indicates that the local environment play an important role at genomic level. Thus the present study shows that the reduction in microalgae in polluted water than the pond water. Variation in restriction fragment length polymorphism could be seen. Hence the Cymbella ventricosa isolated from industrial polluted water had different genomic pattern than the temple pond water.

Conclusion

From this study we conclude that the local environments play an important role in organism's morphological and molecular characters. By this the genomic sequence differences between the same species were identified.

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N. Jagannathan * and K. Murugan (1)

* VELS University, Department of Biotechnology, School of Life Sciences, Chennai-600117, Tamilnadu, India

(1) University of Madras, Gurunanak College, Department of Advance Zoology and Biotechnology, Chennai, India

* Corresponding Author E-mail: jagoilgae@gmail.com
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Author:Jagannathan, N.; Murugan, K.
Publication:International Journal of Biotechnology & Biochemistry
Article Type:Report
Date:Feb 1, 2011
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