Anthony B Blakeney

A simple and rapid preparation of alditol acetates for monosaccharide analysis

Abstract A simple and rapid method is described for the preparation of alditol acetates from monosaccharides. It can be performed in a single tube without transfers or evaporations. Monosaccharides are reduced with sodium borohydride in dimethyl sulphoxide and the resulting alditols acetylated using 1-methylimidazole as the catalyst. Removal of borate is unnecessary and acetylation is complete in 10 min at room temperature. Monosaccharides are quantitatively reduced and acetylated by this procedure. The alditol acetates are completely separated by glass-capillary, gas-liquid chromatography on Silar 10C. The method has been applied to the analysis of monosaccharides in acid hydrolysates of a plant cell-wall.

An improved procedure for the methylation analysis of oligosaccharides and polysaccharides.

An improved procedure for the methylation analysis of oligosaccharides and polysaccharides is described. Steps in the procedure were examined and optimised for quantitative recovery and speed. Methylation was shown to be complete by using [14C]methyl iodide. All operations were performed in the same tube and the need to concentrate solutions containing acetylated alditols of methylated sugars was eliminated, thus minimising losses due to volatilization. The method is convenient, gives high recoveries of acetylated alditols of methylated sugars, and allows analysis of the glycosyl linkages of oligo- or poly-saccharides to be completed within a working day. A wide range of oligo- and poly-saccharides were methylated by this procedure.

The use of microsatellite polymorphisms for the identification of Australian breeding lines of rice (Oryza sativa L.)

Ten microsatellite loci were analysed for 43 cultivars or breeding lines of rice. Polymorphism-Information-Content values ranged from 0.62 to 0.92. The microsatellite markers were found to be useful for cultivar identification and assessment of genetic relationships. Most of the cultivars could be uniquely identified by at least one microsatellite marker. Genetic heterogeneity was detected within rice samples by amplification of microsatellites from DNA extracted from multiple individual plants and also from bulked DNA preparations.

Random amplified polymorphic DNA analysis of Australian rice (Oryza sativa L.) varieties

SummaryThe genetic relationships between rice varieties were analysed by using the polymerase chain reaction (PCR), with arbitrary oligonucleotide primers in the random amplified polymorphic DNA (RAPD) method. PCR with 22 arbitrary primers applied to 37 varieties produced 144 useful markers, of which 67% were polymorphic. Thus, with selected primers sufficient polymorphism could be detected to allow identification of individual varieties. Visual examination of electrophoresis gels and analysis of banding patterns confirmed that commercial Australian and USA lines and their relatives were very closely related, with similarity indices of 88–97%. Three varieties originating from more distant geographical centres were easily distinguished, producing variety-specific amplification profiles and expressing a lower similarity index of 80% to all other varieties tested. PCR offers a potentially simple, rapid and reliable method for rice genotype identification and recognition of lines that could contribute genetic diversity to new commercial varieties.

PCR-based molecular markers for the fragrance gene in rice (Oryza sativa L.).

Abstract The genomic DNA clone RG28, linked to the major fragrance gene of rice (fgr), was assessed for polymorphism in order to produce a PCR-based marker for fragrance. A small mono-nucleotide repeat, that was polymorphic between a pair of fragrant and non-fragrant cultivars, was identified and developed into a co-dominant PCR-based marker. The polymorphism-information-content determinations for three microsatellite markers, that have been genetically mapped near RG28, are also presented. These PCR-based markers will be highly useful in distinguishing fragrance-producing alleles from non-fragrance-producing alleles at the fgr locus.

Detection of neutral and aminosugars from glycoproteins and polysaccharides as their alditol acetates

A new method for the preparation and separation of alditol acetates from neutral sugars has been applied to aminosugars. Reduced aminosugars were rapidly acetylated using 1-methylimidazole as the catalyst without removal of borate formed during reduction. The alditol acetates were separated by glass capillary gas chromatography on Silar 10C. The alditol acetates of aminosugars had retention times much longer than those of neutral sugars. However, the alditol acetates of the deamination products of aminosugars had shorter retention times and were resolved from those of neutral sugars. This method was used for the simultaneous detection of neutral and aminosugars in acid hydrolysates of chitin and the glycoproteins, ovalbumin and peroxidase.

Adsorption of a hydrophobic mutagen to cereal brans and cereal bran dietary fibres

The abilities of brans from the cereals barley, oats, maize, rice, and wheat to adsorb in vitro the hydrophobic, environmental mutagen 1,8-dinitropyrene (DNP) were investigated using a mutagenicity assay. These brans were obtained from known cultivars using defined milling conditions and were chemically characterised. The abilities of total and insoluble dietary fibre preparations obtained from these brans to adsorb DNP were also investigated. The predicted weight of each bran required to adsorb 50% of the added DNP was used to compare the adsorptive abilities of the different brans. The brans were ranked in the order (most effective to least effective): rice, wheat, maize, barley, and oats. The adsorptive abilities of the dietary fibre preparations were not significantly different from the bran from which they were prepared. However, if the dietary fibres (cell walls) were the only components adsorbing the DNP, we would have expected the dietary fibre preparations to have adsorbed more DNP than the equivalent unextracted bran. This suggests that other components, probably starch, also adsorb DNP in the unextracted brans. It is not known why brans from different cereal species differ in adsorptive ability but the lignified cell walls in wheat bran may be important in conferring good adsorptive properties to this bran. The possible relationship between adsorptive ability and ability of the bran from a particular species to protect against colorectal cancer is discussed.

An efficient transformation system for the Australian rice cultivar, Jarrah

A rapid and efficient transformation system for the generation of large numbers of transformed, fertile, transgenic rice (Oryza sativa L.) plants of the Australian rice cultivar, Jarrah, is described. Embryogenic callus pieces derived from mature seeds were bombarded with gold particles coated with DNA. Two plasmids were used, one containing a gene encoding hygromycin phosphotransferase (hph, conferring hygromycin resistance) as a selectable marker and the other containing uidA (gus) as a reporter gene. The calli were selected for their resistance to hygromycin. DNA uptake, integration and expression of the hph and gus gene in selected rice were investigated by various PCR methods and dot blot and Southern analysis of genomic DNA extracted from transformed rice plants. On average one independently transformed hygromycin resistant plant was recovered from every 2.4 pieces of callus bombarded. Selection with Biolaphos using the Bar gene as selectable marker was not successful in this system.

Activity and action pattern of Bacillus licheniformis α-amylase in aqueous ethanol

A purified B. licheniformis α‐amylase in a mixture of ethanol‐aqueous buffer (1:1, ) retains half the activity shown in water alone. In ethanol‐aqueous buffer (7:3, ) about 20% of the activity is retained. The pattern of oligosaccharides produced from amylose changed with ethanol concentration; in aqueous buffer the products are: DP 1 and 2, 33.7%; DP 3, 28.5%; DP 4, 4.4% and DP 5, 33.4%. Whereas in ethanolaqueous buffer (7:3, ) the products are DP 1 and 2, 66.8%; DP 3, 17.3%; DP 4, 4.1 % and DP 5, 11.8%. These results suggest that a change in substrate affinity at the active centre subsites is induced in the ethanolaqueous buffer medium.

Separation of alditol acetates from plasticizers and other contaminants by capillary gas chromatography

Abstract Alditol acetates and contaminating plasticizers were separated on a Silar 10C glass capillary column. The retention times of a range of phthalate, adipate and sebacate esters are reported under chromatographic conditions suitable for the separation both of alditol acetates and of permethylated alditol acetates. Ways of minimizing contamination are discussed.