Bioinformatics in molecular immunology laboratories demonstrated: Modeling an anti-CMV scFv antibody

A scFv (single chain variable fragment) antibody clone from anti-CMV (anti-cucumber mosaic virus) was successfully constructed from immunized mouse and the DNA sequence was submitted to GenBank (AY337618 and AY337619). The expression of a 32 kDa recombinant antibody in bacteria was verified using ELISA (enzyme-linked immunoassay) and western blot. However, elucidation of specific anti-CMV scFv function requires detailed and time consuming immuno-assays. Alternatively, useful functional information on anti-CMV scFV antibody can be obtained using available Bioinformatics tools and techniques without performing tedious assays. Here, we use the commonly used Bioinformatics tools and databases such as BLAST (basic local alignment search tool), GenBank, PDB (protein databank), KABAT numbering, SWISS-MODEL and Insight II to gain specific functional insights into anti-CMV scFv

PLOJDY ANALYSIS AND DNA CONTENT OF MUTANT BANANA "PISANG BERANGAN" USING FLOW CYTOMETRY

Mutagens cause random changes in the nuclear DNA or cytoplasmic organelles, resulting in gene, chromosomal or genomic mutations and hence, create variability. In this study, flow cytometry (FCM) was used to determine ploidy levels and DNA content in gamma-irradiated variants of mutated Pisang Berangan (cv. Intan, AAA) - a local banana genotype. Induced variants such as short plant stature (stunted growth), late flowering plants (late maturity) and abnormalities in bunch characters were selected to study possible changes at the DNA level. The study showed that DNA content of mutated plants differed from non-irradiated control and that irradiation had the most effect at high doses (40 and 60 Gy). The increase of DNA content in 20 Gy and 30 Gy treated plants was not more than that of the control plants. The values of genomic DNA content of gamma-irradiation variants decreased as the dose of irradiation increased from 20 to 60 Gy, indicating that the high dose of gamma-irradiation had a significant effect on the genome of the plants. The analysis further showed that phenotypic variation due to mutagenesis was reflected in the DNA content of the plants. The results also showed that ploidy levels were not affected by gamma-irradiation even at high doses. Keywords: Musa spp./mutation breeding/ flow cytometry/  ploidy level/ DNA conten

CHARACTERIZATION OF MALAYSIAN WILD BANANAS BASED ON ANTHOCYANINS

The male buds of 16 Musa species (Musaceae) populations were investigated by HPLC for the occurrence of anthocyanins. The investigation was based on the presence of 6 anthocyanins. The 16 Musa samples could be classified into three distinct species i.e. Musa acuminata, Musa violascens and Musa balbisiana. Musa acuminata could be divided into two subspecies : malaccensis (lowland) and tmncata (highland) according to their constituents and content of major anthocyanins. No variation was observed in the composition of the anthocyanins of Kedah type ssp. siamea and Selangor types ssp. malaccensis. The classification of M. acuminata into two subspecies based on anthocyanin data further supported the current taxonomic grouping of the species. Key words: Musa acuminata/Musa violascens/Musa balbisiana/Musaceae /HPLC /chemotaxonom

Virus-Specific Read-Through Codon Preference Affects Infectivity of Chimeric Cucumber Green Mottle Mosaic Viruses Displaying a Dengue Virus Epitope

A Cucumber green mottle mosaic virus (CGMMV) was used to present a truncated dengue virus type 2 envelope (E) protein binding region from amino acids 379 to 423 (EB4). The EB4 gene was inserted at the terminal end of the CGMMV coat protein (CP) open reading frame (ORF). Read-through sequences of TMV or CGMMV, CAA-UAG-CAA-UUA, or AAA-UAG-CAA-UUA were, respectively, inserted in between the CP and the EB4 genes. The chimeric clones, pRT, pRG, and pCG+FSRTRE, were transcribed into full-length capped recombinant CGMMV transcripts. Only constructs with the wild-type CGMMV read-through sequence yielded infectious viruses following infection of host plant, muskmelon (Cucumis melo) leaves. The ratio of modified to unmodified CP for the read-through expression clone developed was also found to be approximately 1:1, higher than what has been previously reported. It was also observed that infectivity was not affected by differences in pI between the chimera and its wild counterpart. Analysis of recombinant viruses after 21-days-postinculation (dpi) revealed that deletions occurred resulting in partial reversions of the viral population to near wild type and suggesting that this would be the limiting harvest period for obtaining true to type recombinants with this construct

Total RNA Extraction for the Red Seaweed Gracilaria changii (Gracilariales, Rhodophyta)

Five different RNA extraction methods have been tried out on the red seaweed, Gracilaria changii collected from the mangrove area at Morib, Selangor, Malaysia. Two methods, one utilising guanidinium thiocyanate, and another using cetyltrimethyl ammonium bromide (CTAB), were found to be potential alternatives to obtain pure RNA. By incorporating sand while grinding the tissue, the method using CTAB was found most suitable to obtain pure RNA (high A260:280nm ratio) with high yield (0.16ug RNA per gram of fresh tissue)

Optimisation of RNA Extraction from Gracilaria changii (Gracilariales, Rhodophyta)

RNA extraction from seaweed tissues is problematic due to the presence of polysaccharides and polyphenolic compounds upon cell disruption. Besides, a successful RNA isolation from seaweed tissues can sometimes be strain- and species-specific. Four different methods were used to extract RNA from Gracilaria changii (Gracilariales, Rhodophyta), collected from the mangrove area at Morib, Selangor, Malaysia. An optimised and modified total RNA extraction method was developed for this recalcitrant species. The use of sand in tissue grinding, and the incorporation of phenol extraction at the initial stage resulted in the highest RNA yield (0.65-1.14 microg.g^-1 fresh weight) with high quality (A260:280 ratio 1.80-2.05). The RNA obtained is suitable for cDNA synthesis and future functional genomic studies

Optimization of CTAB-based RNA extraction for in planta Fusarium oxysporum f. sp. cubense gene expression study

A crucial prerequisite for an insightful gene expression study is the quality of the nucleic acid extracted. High-quality nucleic acids allow comparative downstream analyses for both organisms during a phytopathogen infection. However, RNA extraction of pathogen-infected host materials usually involves extraction methods that are optimised individually for either the pathogen or the host. Different sets of buffers or specialised commercial kits are often required. In this study, a streamlined CTAB-based extraction protocol was optimised for both the pure culture of Fusarium oxysporum f. sp. cubense (Foc) and infected banana roots. Foc cultures were grown on PDA overlaid by a nylon membrane and total nucleic acids were successfully extracted from mycelia with a ratio of 100 mg mycelia powder mass to 2 mL of CTAB buffer. Using the optimised protocol, LiCl-precipitated RNAs showed higher values of A260/280 (2.064 ± 0.021) and A260/230 (1.937 ± 0.076) compared to ethanol precipitated RNAs. Similar observation was observed for inoculated banana roots where LiCl-precipitated RNAs showed higher values of A260/280 and A260/230 compared to ethanol precipitated RNAs. qRT-PCR analysis using a pair of Foc specific primers, FoTEF1α, confirmed that the LiCl-precipitated RNA was more suitable for downstream gene expression studies. This extraction protocol is applicable for Foc in planta gene expression study with a high potential to be extended to other filamentous fungal pathogens

Development of a transformation system for Gracilaria changii (Gracilariales, Rhodophyta), a Malaysian red alga via microparticle bombardment

Of the seaweed phycocolloids, agar has the higher price in the world market and is mostly extracted from red seaweeds. Critchley (1993) reported the world market value of agar as US $200 million. Hurtado-Ponce and Umezaki (1988) reported that the world's commercial agar comes mostly from gracilaroids

Identification of molecular markers for disease resistance genes to Fusarium oxysporum f. sp. cubense in Musa acuminata ssp. malaccensis for marker assisted selection (MAS)

Fusarium wilt is a serious soil-borne disease and caused considerable loss to the banana export trade(Rutherford, 1999). Four physiological races of FOC have been reported based on their selective pathogenecity in different banana cultivars. Race 1 had been found virulent on Gros Michel (AAA genotype) while Race 2 on Bluggoe (ABB) and Race 3 on Heliconia. Finally, Race 4 attacking the Cavendish cultivars (AAA) and also virulent on Gros Michel and Bluggoe. Numerous disease control such as soil amendment with Ca or organic matter as well as fumigation with methyl bromide but it only provide temporary solution to this problem. Therefore planting disease resistance bananas is the best approach as it is economical, effective and practical in the long run