Growth characteristics and ion contents of non-selected and salt-selected callus lines of highbush blueberry (Vacdnium corymbosum) cultivars Blue Crop and Denise Blue

Non-selected and sodium chloride selected callus lines of Vacdnium corymbosum L.cv Blue Crop and cv. Denise Blue were grown on media supplemented with 0–100 mM NaCl. For both cultivars, fresh weight and dry weight yields were greater in selected lines on all levels of NaCl. Selected lines of Blue Crop displayed better growth than selected lines of Denise Blue at most concentrations of NaCl. Internal Na⁺ and Cl^– concentrations in selected and non-selected lines of both cultivars increased as external concentration was raised. However, selected lines of Blue Crop and Denise Blue accumulated more Na⁺ and Cl^– than non-selected lines. Selected lines of both cultivars maintained higher levels of K⁺ than non-selected lines on all external NaCl levels. Selected lines of Blue Crop had higher levels of Na⁺ and Cl^– than that of Denise Blue. The results suggest Na⁺ and Cl^– accumulation could be a mechanism allowing better growth in selected lines at moderate salinity levels (50–75 mM NaCl).     

Allele-specific amplification of exon 7 in the survival motor neuron (SMN) genes for molecular diagnosis of spinal muscular atrophy

There are two highly homologous survival motor neuron (SMN) genes in humans but molecular defects in the SMN1 gene cause spinal muscular atrophy (SMA). More than 90% of SMA patients are shown to have a homozygous deletion of exon 7 in the SMN1 gene. Therefore, a simple test for exon 7 deletion would be very useful in the molecular diagnosis of SMA. However, limited methods are available, and most of these methods utilize expensive instruments and consumables. Here, we describe a simple allele-specific PCR test, which can be performed using standard equipment in DNA laboratories. The principle of the test is based on a single nucleotide difference (C versus T) between the exon 7 of SMN1 and SMN2 genes. Using allele-specific primers, two PCR amplifications are performed for each sample to amplify a 404-bp diagnostic fragment, and consequent electrophoresis of PCR products on agarose gel provides definitive information concerning the exon 7 deletion To rule out false negatives, a 500-bp fragment from the N-acetyltransferase gene was coamplified as an internal control in each test. We have, so far, analyzed 41 SMA samples with our method, and tested the validity of results using an independent restriction fragment length polymorphism (RFLP) method. Genotyping results obtained by both methods were in complete agreement for all of the samples analyzed. Our method can also be used to detect heterozygous deletion of exon 7 in SMN genes, if the relative intensities of the diagnostic and internal control bands are determined.     

NeeMDB: Convenient Database for Neem Secondary Metabolites

Indian Neem tree is known for its pesticidal and medicinal properties for centuries. Structure elucidation of large number ofsecondary metabolites responsible for its diverse properties has been achieved. However, this data is spread over various books,scientific reports and publications and difficult to access. We have compiled and stored structural details of neem metabolites inNeeMDB, a database which can be easily accessed, queried and downloaded. NeeMDB would be central in dissipating structuralinformation of neem secondary metabolites world over.     

Optimized conditions for rapd analysis inPinus radiata

Summary Pinus radiata is the most important softwood plantation species in Australia and New Zealand. The improtance of this species in forestry has led to an increasing demand to improve the efficiency of selection time of the production population, which currently takes 13 yr by traditional methods. With the application of molecular biology techniques such as random amplified polymorphic DNA (RAPD) the selection period can be reduced to 6 yr. In this study, the conditions for RAPD were optimized and the feasibility of this marker system was investigated with different families ofPinus radiata from Tasmania and South Australia. Best concentrations of Taq-polymerase (1 U), magnesium chloride (2 mM), and template DNA (20 ng) were selected to test different polymerase chain reaction (PCR) thermocycler profiles. Devey's et al. (1996) program was the most effective for production of clear RAPD bands. Best conditions were investigated to screen 10–12 bp arbitrary Breasatec and Operon primers. Both types were found useful at detecting genetic variation between families. Seventy percent and thirty percent of the selected Bresatec and Operon primers, respectively, produced polymorphic bands.     

Isolating conifer DNA: A superior polysaccharide elimination method

Genomic DNA was isolated from frozen needles of maturePinus radiata clones using a modified extraction technique incorporating cetyltrimethylammonium bromide (CTAB) for cell lysis. A high sodium chloride concentration (2 M) was used at 2 stages of the extraction procedure to eradicate polysaccharides, yielding pure genomic DNA suitable for restriction enzyme digestion and PCR amplification. Extractions were scaled down to suit 1.5-mL Eppendorf tubes, allowing easier handling and enhanced sterility.     

M13-based genotyping of marine cyanobacterial strains from the Indian subcontinent and maintained in the NFMC germplasm collection

The aim of this study was to analyze marine cyanobacterial culture collections strains of the Indian subcontinent at the level below species. This is important to improve the abilities of service culture collections to provide their user community with correctly identified and clean organisms. A total of 50 marine cyanobacterial strains were genotyped with M13 polymerase chain reaction (PCR) fingerprinting to provide diagnostic fingerprints for each culture. Depending on the strains, 9 to 26 bands were observed for the primer tested. Within the species, strains representing different isolates were genetically clearly different. Data obtained from genomic fingerprinting were used to construct binary distance matrix, and the neighbor-joining tree constructed demonstrated the ability of this method to differentiate strains at the intraspecific level. An important and useful result obtained in this study is the application of the M13 PCR fingerprinting method on almost all forms of cyanobacteria for strain and species discrimination.     

Optimizing conditions for DNA isolation fromPinus radiata

Summary Genomic DNA was isolated fromin vitro Pinus radiata seedling with five DNA isolation protocols commonly used for pines. The methods described by Jobes et al. (1995) and Nelson et al. (1994) utilize sodium dodecyl sulfate, whereas those of Murray and Thompson (1980), Doyle and Doyle (1990), and Devey et al. (1996) use cetyltrimethyl ammonium bromide for cell lysis. The quality and quantity of the isolated DNA was measured and compared. Lithium chloride was found to be more effective than RNase for minimizing the amount of RNA present in the solution. Protocols described by Jobes et al. (1995) and Devey et al. (1996) yielded a large quantity of pure DNA which was suitable for restriction enzyme digestion and polymerase chain reaction amplification. With these methods, 37 to 79 μg of DNA with an A_260/280 ratio between 1.7 and 1.9 was obtained from 1 g ofPinus radiata seedlings grownin vitro.     

Comparison of bovine RNA polymerase III promoters for short hairpin RNA expression

RNA interference (RNAi) mediated by DNA-based expression of short hairpin RNA (shRNA) is a powerful method of sequence-specific gene knockdown. A number of vectors for expression of shRNA have been developed that feature promoters from RNA polymerase III (pol III)-transcribed genes of mouse or human origin. To advance the use of RNAi as a tool for functional genomic research and for future development of specific therapeutics in the bovine species, we have developed shRNA expression vectors that feature novel bovine RNA pol III promoters. We characterized two bovine U6 small nuclear RNA (snRNA) promoters (bU6-2 and bU6-3) and a bovine 7SK snRNA promoter (b7SK). We compared the efficiency of each of these promoters to express shRNA molecules. Promoter activity was measured in the context of RNAi by targeting and suppressing the reporter gene encoding enhanced green fluorescent protein. Results show that the b7SK promoter induced the greatest level of suppression in a range of cell lines. The comparison of these bovine promoters in shRNA expression is an important component for the future development of bovine-specific RNAi-based research.