MOSP - Macao Scientific Publishers

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FLAG tag module for PCR based gene targeting

Rajeev Ranjan, Sumit Kumar Verma and Shakil Ahmed*

Abstract: Epitope tagging of yeast proteins has become an efficient tool for biochemical analysis of protein of interest. The epitope-tagged proteins can be used for western blotting, immunoprecipitation and immunofluorescence experiments without the need to raise specific antibodies, thus saving considerable time and expense. We have constructed plasmid containing FLAG tag with kanMX6 module, which allows selection of G418-resistant cells in yeast. The same set of primers that amplify module constructed by Bahler et al. (1998) can be used to amplify the FLAG tag module constructed in this study. The linear DNA fragment containing FLAG tag module with flanking homology region of gene of interest can be efficiently integrated on the yeast genome, using homologous recombination. We have successfully FLAG tag wat1/pop3 gene at its chromosomal locus and confirmed by western blot analysis. This construct can be very useful for generating C terminal tagging of desired genes at its normal chromosomal locus without interfering with their function.[...] Read More.

Keywords: S. pombe, epitope tagging, FLAG tag, pFA6a plasmid, wat1/ pop3.

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Identification of a restriction endonuclease (SacC1) from Saccharomyces cerevisiae

Mukaram Shikara

Abstract: SacC1 is a novel restriction endonuclease from Saccharomyces cerevisiae that recognizes the palindromic sequence 5’CTCGAC3’ cleaving both DNA strands upstream and downstream of its recognition sequence and makes a staggered cut at the distance of five bases from the recognition sequence on the upper strand and at the seventh base on the complementary strand. It shares similar characteristics with Sac I from Streptomyces achromogenes as well as Sst1 from Streptomyces Stanford and Psp124B1 from Pseudomonas species. It has been purified by ammonium sulphate precipitation, dialysis, and gel filtration using phosphocellulose, DEAE-cellulose and Sephadex G-100 with an optimal pH range (7.5-8.5), active at 37°C and dependent on Mg +2 or Mn2+ which increases its activity by 4- and 2-folds, respectively, while other cations decrease its activity to some extents. Cleavage on both sides of the recognition sequence is characteristic of Type IIB systems but all IIB enzymes studied so far have been found to recognize discontinuous sites and a distinctive subunit/domain organization that is not present in the SacC1 enzyme. There are similarities between SacC1 and other homing endonucleases belonging to the LAGLIDADG family such as a requirement for Mg2+ (or Mn2+) for cleavage to take place, optimal activity at alkaline pH and stimulation of the reaction by moderate concentrations of the monovalent cation.[...] Read More.

Keywords: Purification, recognition site, restriction enzyme, Saccharomyces, Streptomyces, Type IIB.

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Improved antimicrobial activity of the Tanzanian edible mushroom Coprinus cinereus (Schaeff) Gray by chicken manure supplemented solid sisal wastes substrates

Liberata Nyang’oso Mwita, Anthony Manoni Mshandete and Sylvester Leonard Lyantagaye*

Abstract: The Tanzanian edible mushroom species Coprinus cinereus was grown on sisal waste substrates supplemented with chicken manure with the aim to evaluate the effects of the chicken manure supplement on the antimicrobial activity of the mushroom’s extracts. Crude ethyl acetate extracts were prepared from the mushroom’s fruiting bodies harvested at pre-capping, capping and post capping stages, and the extracts were tested for antimicrobial activity, using the agar well method. The antimicrobial activity was observed only in capping and post capping stages of the mushrooms and the activity generally increased with increased percentage of manure supplementation. These findings show that Tanzanian edible C. cinereus mushroom contains antimicrobial compounds and chicken manure could be used in the cultivation of the mushroom to increase the production of active secondary metabolites, which could be used as lead compounds for discovery of new and more effective drugs against microbial infections.[...] Read More.

Keywords: Coprinus cinereus, antimicrobial, chicken manure.

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Mycotic contamination of stockfish sold in Jos, Nigeria

S. A. Junaid*, F. Olarubofin and A. O. Olabode

Abstract: This study was aimed at isolating and identifying the fungi associated with stockfish contamination in Jos Metropolis. A total of 100 stockfish samples were randomly purchased from four markets namely, Terminus, Kwararafa, Katako and Gada biu in Jos town, Plateau State, Nigeria. The stockfish samples were assayed for fungal contamination and moisture content using standard procedures. All the stockfish samples were contaminated with fungi. Seven different fungi were found to be associated with the stockfish samples sold in the four different markets. The associated fungi were Mucor Spp, Asergillus flavus, Trichophyton verrucosum, Aspergillus niger, Aspergillus fumigatus, Penicillin Spp and Rhizopus Spp. It was observed that Mucor Spp had the highest rate of occurrence among the isolated fungi. The moisture content was between 6 - 27%. Results from the study are useful in developing and establishing public health standards as consumption of these fungi exposes the consumers to the probable toxic metabolites produced by the fungi.[...] Read More.

Keywords: Mycotic contamination stockfish, cod.

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Determination of effective dose of garlic for controlling seedborne fungal disease of tomato

M. M. Rashid, A. B. M. Ruhul Amin and F. Rahman

Abstract: Tomato seeds collected from farmer’s were treated with garlic tablet at concentration 1:3 w/v, 1:4 w/v, 1:5 w/v and 1:6 w/v for controlling seedborne fungal diseases. Seed health status of treated seeds was evaluated following blotter incubation method. Three different fungal pathogens viz., Aspergillus spp., Fusarium spp. and Penicillium spp. were identified from seed samples by blotter incubation method. Garlic tablet at 1:3 w/v dose showed better performance in increasing seed germination and reducing prevalance of fungal pathogens over control treatment. The highest germination recorded was 71.25% at 1:3 w/v dose of garlic tablet which represents an increase of 11.25% over control. Germination percentage was increased 18.75% over control when treated seeds were sown in tray soil. Substantial importance in seed quality was noticed in reducing hard seed, damping off, blighted seedlings and tip over. In pot experiment, 1:3 w/v dose also performed best to yield the lowest percentage of hard seed, damping off, blighted seedlings, tip over and seedlings with highest seed germination.[...] Read More.

Keywords: Garlic, tomato, seed treatment, germination, seedborne pathogens, Aspergillus, Fusarium, Penicillium.

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Effects of storage conditions and tillage operation on some fungal diseases and yield of maize

G. O. Ihejirika*, M. I. Nwufo and S. O. Anagboso

Abstract: Maize (Zea mays L) belongs to the family Poaceae and it is mostly grown as food for man and feed for animal. A two-season experiment was conducted in 2006 and 2007, respectively, to determine the effect of storage condition and tillage operation on some fungal diseases and yield of maize. Analysis of variance indicated that storage condition significantly affected plant height (cm) 5.69; 6.26 at 9 weeks after planting. Tillage operation was statistically significant on leaf spot and blight infection at 9 weeks after planting at 5% probability level. Interaction of storage condition and tillage operation was also significant on blight infection at 9 weeks after planting 0.50; 0.58. Seeds from dehusked maize recorded highest plant height, leaf formation and grain yield, while shelled was lowest on both parameters in 2006 and 2007, respectively. However, the three storage conditions investigated were statistically similar on blight infection. Dehusked and undehusked plots recorded statistically similar result on leaf spot severity while shelled had the highest. Spot tilled plots recorded lowest leaf spot and blight severity but highest grain yields when zero-tilled plots (control) had the highest in all the field diseases investigated, but lowest grain yield in 2006 and 2007 respectively. Micro-organisms identified on infected leaves were Helminthosporium spp, Spermospora spp, while Fusarium spp, Penicilium spp, Blastomyces and Aspergillus species were identified with infected grains with Fusarium and Penicilium species occurring highest in 2006 and 2007, respectively.[...] Read More.

Keywords: Storage condition, tillage operation, fungal diseases, yield, maize.

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Influence of bacteria and protozoa from the rumen of buffalo on in-vitro activities of anaerobic fungus Caecomyces sp. isolated from the feces of elephant

Ravinder Nagpal*, Anil Kumar Puniya, Jatinder Paul Sehgal and Kishan Singh

Abstract: Anaerobic fungal isolates Caecomyces sp. from the feces of elephant and Orpinomyces sp. from buffalo rumen were co- cultured in-vitro with rumen bacterial and protozoal fractions collected from buffalo to observe the possible fate of these fungi in the rumen, if inoculated as microbial-feed supplements. When co-cultured together or separately with rumen bacteria and protozoa, Caecomyces sp. was adversely affected. However, bacterial and protozoal counts were higher, compared to the counts when grown alone. Similar patterns of results were observed when Orpinomyces sp. was grown in co-culture with bacteria and protozoa separately as well as together, indicating that it is possibly the inhibitory action of bacteria and protozoa, and not inter-species competition, that affects the growth of fungi preventing them from attaining their full fibre-degrading potential. Conversely, although fungal counts were lowered during their co-culturing with bacterial and protozoal fractions, their co-culturing increased the FPase activity of the co-cultured fraction which could be the apparent reason for enhanced fibre degradation.[...] Read More.

Keywords: Rumen microflora, anaerobic fungi, microbial interactions, fibre degradation.

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Overview on Echinophora platyloba, a synergistic anti-fungal agent candidate

Avijgan Majid*, Mahboubi Mohaddesse, Darabi Mahdi, Saadat Mahdi, Sarikhani Sanaz and Nazilla Kassaiyan

Abstract: Echinophora Platyloba DC. is one of the four native species of this plant in Iran. The aim of this five-step study was to investigate the antimicrobial properties of this plant. Dried aerial parts of the plant were extracted by ethanol 70% in percolator. The antimicrobial activity of ethanolic extract was evaluated against dermatophytes, Candida (C) albicans and gram positive bacteria by agar dilution method and microbroth dilution assay. Finally, the synergistic effect of Amphotericin B plus 5% ethanolic extract against C. albicans was determined by measuring MIC (minimum inhibitory concentration) and MLC (minimum lethal concentration) values. Gram positive bacteria were resistant to the extract according to measurement of zones of inhibition; Trichophyton schenlaini and Trichophyton verucosum were sensitive to concentrations ³ 35 mg/ml, while other dermatophytes showed various susceptibilities to extract. MIC value of 5% ethanolic extract was 2 mg/ml against C. albicans using broth micro dilution method. In synergism assay, there was a 50% reduction in MIC and a 75% reduction in MLC values of the mixture of Amphotericin B and 5% ethanolic extract against C. albicans in comparison to Amphotericin B alone. Regarding this study, some degrees of synergy was recorded in the combination of Amphotericin B plus E. platyloba extract covering C. albicans which represented promising finding in antifungal therapy.[...] Read More.

Keywords: Amphotericin B, Candida albicans, Echinophora platyloba, ethanolic extract, traditional medicine.

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Some sugar fungi in coastal sand dunes of Orissa, India

T. Panda

Abstract: Occurrence and distribution of sugar fungi was studied from soil and leaf litter in coastal sand dunes of Orissa for a period of two years covering three distinct seasons. Fungal succession of litter was also studied. Microbial isolation and soil analysis was performed using standard procedures. Maximum population density was observed in the rainy season followed by winter and lastly summer. Higher microbial populations were encountered in plantation soil than the barren sand. They corresponded to the fluctuation of prevailing temperature, moisture and total organic carbon content of the said habitat. A total of 8 species of sugar fungi were isolated of which soil and the leaf litter had a share of 8 species each. Maximum population of sugar fungi was recorded from coastal sand dunes with Casuarina plantation which can be due to less competition with other fungi.[...] Read More.

Keywords: Sugar fungi, coastal sand dune, fungi, leaf litter.

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Antifungal properties of essential oils and some constituents to reduce foodborne pathogen

Yaouba Aoudou, Tatsadjieu Ngouné Léopold*, Jazet Dongmo Pierre Michel, Etoa François Xavier and Mbofung Carl Moses

Abstract: Investigations were conducted to evaluate the antifungal activities of the essential oils of Lippia rugosa, Plectranthus glandulosus, Clausena anisata and Vepris heterophylla and some essential oils compounds as citral, geraniol, nerol, citronellol, fenchone, linalool, 1,8 cineol, nerolidol, terpen 4-ol and -terpinolene on mycelia growth of different strains of Aspergillus, Penicillium and Fusarium genus, common fungi causing spoilage of stored food product. The disc diffusion method was used to evaluate fungal growth inhibition at various concentrations. The strains of fungi exhibited similar susceptibilities (90 mm) to the action of L. rugosa essential oil and different susceptibilities (34 - 90 mm) to P. glandulosus, 0 to 44 mm to C. anisata and 0 to 30 mm to V. heterophylla essential oils. These inhibition halos varied from 0 to 84 mm according to each compound activity. The mycelial growth of fungal species tested was totally inhibited by MIC values ranged from 0.2 to 0.6 mg/ml for L. rugosa, 0.8 to 2 mg/ml for P. glandulosus and 0.5 to 1.2 mg/ml for citral, geraniol, nerol and citronellol which are the more active among the ten components tested. Results obtained indicate the possibility of exploiting L. rugosa, P. glandulosus essential oils and citral, geraniol, nerol, and citronellol to fight these strains responsible for biodeterioration of stored food.[...] Read More.

Keywords: Sanitizing agents, essential oils, antifungal activity, essential oil constituents.

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