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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, www.discovery.org.inhttp://www.discovery.org.in/ijs.htm © 2012 discovery publication. All rights reserved

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Vishwesh R. Potkar*, Pratima S. JadhavDepartment 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, E-mail: [email protected], Contact- (+91) 9029960082

Received 28 September; accepted 19 October; published online 01 November; printed 16 November 2012

ABSTRACTGenomic DNA was isolated from the Alternaria alternata. ITS region was amplified using universal primers and sequenced. 5.8 S gene found to be highlyconserved and length found 159 bp and GC% was 44.7. In the present study the RNA secondary structure was predicted using program MulFold version2.0 (Zuker, 1989). The predicted secondary structure for a functional 5.8S gene, with two large central loops from which four helices emerge. The DGrequired 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 thatthose motifs play an important biological role in rRNA function.

1. INTRODUCTIONAlternaria is a genus of ascomycete fungi. Alternaria species are known as major plant pathogens. They are also common allergens in humans, growingindoors and causing hay fever or hypersensitivity reactions that sometimes lead to asthma. They readily cause opportunistic infections inimmunocompromised 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 orcylindrical beak and have a pale brown, smooth walled orverrucose. However, the genetic makeup, environment and fieldmanagement practices influence the morphology of thepathogen (Simmons, 1985), limiting the accuracy ofmorphological pathogen characterization. This creates difficultyin delineating or delimiting isolates as well as identifying new orrare pathogen species that they may be involved. An example ofsuch occurrence could be A.triticina, with dimensions similar toA. alternata which has only been reported in India (Rotem,1994). At least 20% of agricultural spoilage is caused byAlternaria species. Many human health disorders can be caused by these fungi, which grow on skin and mucous membranes, including on the eyeballsand 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 upperrespiratory infections in AIDS patients, asthma in people with sensitivity, and has been implicated in chronic rhinosinusitis. Alternaria spp. grows rapidlyand the colony size reaches a diameter of 3 to 9 cm following incubation at 25°C for 7 days on potato glucose agar. The colony is flat, downy to woollyand is covered by grayish, short, aerial hyphae in time. The surface is greyish white at the beginning which later darkens and becomes greenish black orolive 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 widelyused 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 ofthe initial transcript play important roles in ribosome assembly (Lalev & Nazar 1999; Lalev et al. 2000; Lalev & Nazar 2001), and putative secondarystructures 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 theinternal spacers as well (Wolf et al. 2005). Characters from nuclear ribosomal gene sequences have been used for hypotheses of phylogeneticrelationships among even distantly related organisms. Molecular phylogenetic analyses require the alignment of homologous sequence characters, andguidance 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, andeukaryotic organelles (Cedergren et al. 1988). A further application of structure information to phylogenetics is recoding structure into new ITS regionshave 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 weremere ‘junk DNA’, whose evolution resulted from the accumulation of chance mutations unfettered by any functional constraints. However, research onplants 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), resizingchanges (mutations increasing or decreasing feature sizes) or silent changes (mutations not altering the secondary structure). These trends cannot yet bedescribed as directional, and the timing of putative events relative to clade separation is unknown (an event might be suggested by a homoplasiousnucleotide character or we might miss data to correctly infer an event’s occurrence). This precludes two useful future uses of pointing out such

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

Predicted secondary structure of 5.8S gene in Alternaria alternata

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Figure 1Internal transcribed spacer region of ribosomal RNA

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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 ofa functional ribosome.

2. MATERIAL AND METHODSThe isolate cultures were maintained on potato dextrose agar (PDA). Fungal cultures were raised by using actively growing fungal plugs from mothercultures 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 laboratorybenches 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 SequencingWhole-cell DNA was isolated from Alternaria alternata by the Chelex method (Walsh et al., 1991; Hirata and Takamatsu, 1996). Primer pairs ITS1 (50-TCC 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 internaltranscribed spacer (ITS) 1, the 5·8S gene and the ITS2 regions (Jasalavich et al., 1995). PCR reactions were conducted in 50 μl volumes as previouslydescribed (Hirata and Takamatsu, 1996). A negative control lacking template DNA was included for each set of reactions. The PCR product wassubjected to preparative electrophoresis in 1.5% agarose gel in TAE buffer. The DNA product of each amplification was then excised from the ethidiumbromide-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 fromGenBank at the National Institutes of Health.

3. RESULTS AND DISCUSSIONITS 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 188bp 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 gene5.8S 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 partof the 45S precursor that also contains 18S and 28S rRNA. Itsfunction is thought to be in 5.8S rRNA ribosome translocation. It isalso known to form covalent linkage to the p53 tumour suppressorprotein. 5.8S rRNA is also found in archaea.The internaltranscribed spacer regions and the 5.8S rDNA were definedbased on the conserved sequence at the 3’end of the 18S gene,the 5’ and 3’ ends of the 5.8S gene, and the 5’ end of the 26Sgene (Hausner & Wang 2005). Secondary structure of 5.8S rDNAsequence was reconstructed under specific settings for basepairing. The stem loop structures were folded using the mfold webserver (http://mfold.rna.albany.edu/) Zuker 2003. Maximization ofthe hydrogen bonding forming solid stems, and the largestnegative 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. TheDG required for formation of the secondary structure of the 5.8Sgene 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 conservedamong angiosperms: M1 (5’-CGAUGAAGAACGUAGC-3’) andM3 (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 (5’-CGATGAAGAACGCAGC-3’), M2 (5’-GAATTGCAGAATTC -3’),and M3 (5’-TTTGAACGCA- 3’). At the DNA level, the motif M2harbors an EcoRI restriction site (underlined), which is highlyconserved in fungi and distinguishes between fungal andangiosperm M2 motifs (Jobes and Thien, 1997). which suggestthat those motifs play an important biological role in rRNAfunction.

The nucleotide sequences of the ITS regions were highlyvariable among powdery mildew species, while relativelyconserved regions were also present. It has been reported thatthe ITS regions as well as rRNA coding regions form secondarystructures, which function in the maturation of rRNA precursors inyeast (Raué and Planta, 1995). We therefore calculated thesecondary structures using the computer programs MulFold. 5.8SrRNA can be used as a reference gene for miRNA detection.

ACKNOWLEDGEMENTAuthors are grateful to the Director, Institute of Science, Mumbai, India.

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Figure 2Secondary Structure of 5.8S gene in Alternaria alternata

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