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RESEARCH

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Indian Journal of Science, Volume 1, Number 1, November 2012

EISSN 2319 7749

ISSN 2319 7730

Science
Predicted secondary structure of 5.8S gene in Alternaria alternata
Vishwesh R. Potkar*, Pratima S. Jadhav
Department of Biochemistry, Institute of Science, Mumbai - 400 032, India *Correspondence to: Vishwesh R. Potkar, Department of Biochemistry, Institute of Science, 15, Madam Cama Road, Fort, Mumbai Mumbai - 400 032, India, Email: vishweshpotkar@gmail.com, Contact- (+91) 9029960082 Received 28 September; accepted 19 October; published online 01 November; printed 16 November 2012

Indian Journal of

Genomic DNA was isolated from the Alternaria alternata. ITS region was amplified using universal primers and sequenced. 5.8 S gene found to be highly conserved and length found 159 bp and GC% was 44.7. In the present study the RNA secondary structure was predicted using program MulFold version 2.0 (Zuker, 1989). The predicted secondary structure for a functional 5.8S gene, with two large central loops from which four helices emerge. The DG required for formation of the secondary structure of the 5.8S gene was -39.25 kcal/mol. At the DNA level, the motif M2 harbors an EcoRI restriction site (underlined), which is highly conserved in fungi and distinguishes between fungal and angiosperm M2 motifs (Jobes and Thien, 1997) which suggest that those motifs play an important biological role in rRNA function.

ABSTRACT

1. INTRODUCTION

Alternaria is a genus of ascomycete fungi. Alternaria species are known as major plant pathogens. They are also common allergens in humans, growing

indoors and causing hay fever or hypersensitivity reactions that sometimes lead to asthma. They readily cause opportunistic infections in immunocompromised people such as AIDS patients. Alternaria alternata, was identified as the causal agent of stem and leaf blight on sweet potato (Lenne, 1991, Clark and Moyer, 1988). According to description by David (1991), conidia of A. alternata are sometimes referred to as obclative, obpyriform, ovoid or ellipsoidal often with a short conical or cylindrical beak and have a pale brown, smooth walled or verrucose. However, the genetic makeup, environment and field management practices influence the morphology of the pathogen (Simmons, 1985), limiting the accuracy of morphological pathogen characterization. This creates difficulty in delineating or delimiting isolates as well as identifying new or rare pathogen species that they may be involved. An example of Figure 1 such occurrence could be A.triticina, with dimensions similar to Internal transcribed spacer region of ribosomal RNA A. alternata which has only been reported in India (Rotem, 1994). At least 20% of agricultural spoilage is caused by Alternaria species. Many human health disorders can be caused by these fungi, which grow on skin and mucous membranes, including on the eyeballs and within the respiratory tract. Allergies are common, but serious infections are rare, except in people with compromised immune systems. However, species of this fungal genus are often prolific producers of a variety of toxic compounds. Alternaria alternata causes early blight of potato, Leaf spot disease in Withania somnifera and can infest many other plants. It also causes upper respiratory infections in AIDS patients, asthma in people with sensitivity, and has been implicated in chronic rhinosinusitis. Alternaria spp. grows rapidly and the colony size reaches a diameter of 3 to 9 cm following incubation at 25C for 7 days on potato glucose agar. The colony is flat, downy to woolly and is covered by grayish, short, aerial hyphae in time. The surface is greyish white at the beginning which later darkens and becomes greenish black or olive brown with a light border. The reverse side is typically brown to black due to pigment production (Collier et al 1998). rRNA genes have been widely used in systematic studies in fungi and beyond, and are common targets for identifying and quantifying phylotypes in medical and environmental samples. The coding (we use coding as coding for RNA) SSU rRNA and LSU rRNA genes are highly conserved. Evidence suggests that secondary structures of the initial transcript play important roles in ribosome assembly (Lalev & Nazar 1999; Lalev et al. 2000; Lalev & Nazar 2001), and putative secondary structures have long been recognized and archived for the coding regions of rDNA (van de Peer et al. 2000; Cannone et al. 2002), and recently for the internal spacers as well (Wolf et al. 2005). Characters from nuclear ribosomal gene sequences have been used for hypotheses of phylogenetic relationships among even distantly related organisms. Molecular phylogenetic analyses require the alignment of homologous sequence characters, and guidance from secondary structure information may aid in the alignment of homologous regions for phylogenetic analysis among plant and animal species (Jobes & Thien 1997; Goertzen et al. 2003; Xia et al. 2003), even between genomes as evolutionarily distant as of eukaryotic nuclei, prokaryotes, and eukaryotic organelles (Cedergren et al. 1988). A further application of structure information to phylogenetics is recoding structure into new ITS regions have been used for phylogenetic analyses at the species to generic level, yet their primary nucleotide sequence often contains insertions and deletions (indels) making alignment difficult much beyond infraspecific levels. This ITS variability led to the assumption that non-coding ITS1 and ITS2 regions were mere junk DNA, whose evolution resulted from the accumulation of chance mutations unfettered by any functional constraints. However, research on plants and green algae suggested that ITS rDNA sequences provide evidence at a super-generic level (Baldwin et al. 1995; Hershkovitz & Zimmer 1996; Mai & Coleman 1997) and contain diagnostic characters for deeper divergences (Hershkovitz & Lewis 1996). Mutational events include compensatory base changes, one-sided changes (mutations leading to introduction or loss of new features), resizing changes (mutations increasing or decreasing feature sizes) or silent changes (mutations not altering the secondary structure). These trends cannot yet be described as directional, and the timing of putative events relative to clade separation is unknown (an event might be suggested by a homoplasious nucleotide character or we might miss data to correctly infer an events occurrence). This precludes two useful future uses of pointing out such
Vishwesh R. Potkar et al. Predicted secondary structure of 5.8S gene in Alternaria alternata, Indian Journal of Science, 2012, 1(1), 51-53, http://www.discovery.org.in/ijs.htm

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hypothetical events: (1) possible confirmation with independent data; and (2) possible experimentation on the feasibility of such event to allow formation of a functional ribosome.

2. MATERIAL AND METHODS

The isolate cultures were maintained on potato dextrose agar (PDA). Fungal cultures were raised by using actively growing fungal plugs from mother cultures with a sterile scalpel and then placed on fresh PDA media (39 g of PDA/ 1 liter of distilled water. The cultures were then placed on laboratory benches and left to grow for about 7-10 days or until there was enough mycelia to harvest for DNA extraction.

2.1. Isolation of DNA, PCR and Sequencing

Whole-cell DNA was isolated from Alternaria alternata by the Chelex method (Walsh et al., 1991; Hirata and Takamatsu, 1996). Primer pairs ITS1 (50TCC GTA GGT GAA CCT GCG- 30) and ITS4 (50-TCC TCC GCT TAT TGA TAT GC-30) were used for PCR amplification of rDNA containing the internal transcribed spacer (ITS) 1, the 58S gene and the ITS2 regions (Jasalavich et al., 1995). PCR reactions were conducted in 50 l volumes as previously described (Hirata and Takamatsu, 1996). A negative control lacking template DNA was included for each set of reactions. The PCR product was subjected to preparative electrophoresis in 1.5% agarose gel in TAE buffer. The DNA product of each amplification was then excised from the ethidium bromide-stained gel. ITS sequences of all species were obtained using primers and sequencing was carried out on ABI Sequencer (Chromous Biotech, Bangalore) with minor manual adjustments. Each ITS DNA sequence was compared by using the BLAST alignment program with data available from GenBank at the National Institutes of Health.

3. RESULTS AND DISCUSSION

ITS sequence of Alternaria alternata was uploaded in NCBI genbank with accession number JX154674.1. The length of entire ITS regions and GC % were calculated by using online bioinformatics tools. ITS length was found 570 base pairs (bp) where as GC% was 46. Length of ITS1 and ITS2 were 188 bp and 223 bp respectively whereas GC% was 43.6 and 48.9 respectively. 5.8 S gene found to be highly conserved and length found 159 bp and GC% was 44.7.

3.1. Secondary structure analysis of 5.8S gene

5.8S ribosomal RNA (5.8S rRNA) is a non-coding RNA component of the large subunit of the eukaryotic ribosome and so plays an important role in protein translation. It is transcribed by RNA polymerase I as part of the 45S precursor that also contains 18S and 28S rRNA. Its function is thought to be in 5.8S rRNA ribosome translocation. It is also known to form covalent linkage to the p53 tumour suppressor protein. 5.8S rRNA is also found in archaea.The internal transcribed spacer regions and the 5.8S rDNA were defined based on the conserved sequence at the 3end of the 18S gene, the 5 and 3 ends of the 5.8S gene, and the 5 end of the 26S gene (Hausner & Wang 2005). Secondary structure of 5.8S rDNA sequence was reconstructed under specific settings for base pairing. The stem loop structures were folded using the mfold web server (http://mfold.rna.albany.edu/) Zuker 2003. Maximization of the hydrogen bonding forming solid stems, and the largest negative delta g value (free energy). dG found is -39.25 kcal/mol The predicted secondary structure for a functional 5.8S gene, with two large central loops from which four helices emerge. The DG required for formation of the secondary structure of the 5.8S gene was -39.25 kcal/mol, and the CG content was 44.7%. Conserved motifs for the 5.8S gene are poorly described in fungi. However, at least three motifs of the 5.8S gene are conserved among angiosperms: M1 (5-CGAUGAAGAACGUAGC-3) and M3 (5-UUUGAACGCA-3) (Harpke and Peterson, 2008), and M2 (5-GAAUUGCAGAAUCC-3) (Jobes and Thien, 1997). In A. alternata, those three motifs were found as M1 (5CGATGAAGAACGCAGC-3), M2 (5-GAATTGCAGAATTC -3), and M3 (5-TTTGAACGCA- 3). At the DNA level, the motif M2 harbors an EcoRI restriction site (underlined), which is highly conserved in fungi and distinguishes between fungal and angiosperm M2 motifs (Jobes and Thien, 1997). which suggest that those motifs play an important biological role in rRNA function. The nucleotide sequences of the ITS regions were highly variable among powdery mildew species, while relatively conserved regions were also present. It has been reported that the ITS regions as well as rRNA coding regions form secondary structures, which function in the maturation of rRNA precursors in yeast (Rau and Planta, 1995). We therefore calculated the Figure 2 secondary structures using the computer programs MulFold. 5.8S Secondary Structure of 5.8S gene in Alternaria alternata rRNA can be used as a reference gene for miRNA detection.

ACKNOWLEDGEMENT REFERENCES
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Authors are grateful to the Director, Institute of Science, Mumbai, India.


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Vishwesh R. Potkar et al. Predicted secondary structure of 5.8S gene in Alternaria alternata, Indian Journal of Science, 2012, 1(1), 51-53, http://www.discovery.org.in/ijs.htm

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Vishwesh R. Potkar et al. Predicted secondary structure of 5.8S gene in Alternaria alternata, Indian Journal of Science, 2012, 1(1), 51-53, http://www.discovery.org.in/ijs.htm

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