R. P. Johari

Characterization and Cloning of ODAP Degrading Gene from a Soil Microbe

The strain BYT-1, capable of utilizing ODAP/DAP as a sole source of nitrogen and carbon was identified as Psuedomonas stutzeri by various microbial and biochemical tests. Transformation experiments showed that the ODAP utilizing property Is encoded by the plasmid. Restriction of plasmid DNA with Pstl, followed by cloning of fragments and screening of ODAP containing medium, led to the isolation of a clone with insert size of −3.3 kb, which encoded ODAP metabolizing property. The growth and ODAP/DAP utilization by this clone (TB) was almost similar to that of the wild type strain.

Cloning and Expression of OX-DAPRO Degrading Genes from Soil Microbe

The strain BYK 1, capable ot utilizing DAP or OX-DAPRO as a sole source ot nitrogen and carbon was Identitied as Pseudomonas stutzeri by various microbial and biochemical tests. Transtormation experiments showed that the OX-DAPRO utilization property Is encoded by the plasmid. Restriction of the plasmid DNA toll owed by cloning ot fragments and screening on OX-DAPRO medium led to Isolation of a clone with DNA Insert of ∼ 2.2 kb (designated as BSKS) which encoded OX-DAPRO degrading gel)e. The growth and OX-DAPRO utilization with this fragment was similar to wild type strain.

Molecular Characterization of Glutenins in Wheat Varieties Differing in Chapati Quality Characteristics

Five wheat (Triticum aestivum) varieties differing in chapati quality characteristics viz. C-306, K-68, HD-2745 and HD-2735 with good and Sonalika with poor chapati quality characteristics, were selected for the characterization or distribution of glutenin genes. Polymorphism was observed when genomic DNA of wheat varieties was hybridized with a HMW glutenin probe [glutenin subunit 10 (Dy10)]. No hybridization was observed in Sonalika. PCR amplification of genomic DNA with the LMW glutenin gene-specific primers did not show any polymorphism. However, with HMW glutenin gene-specific primers a single band of ∼ 650 by was obtained in all the good chapati characteristic wheat varieties.The amplified fragment was sequenced and found to have sequence homology with HMW glutenin subunit Dx5.The deduced protein structure analysis showed that the peptide was made up of N-terminally placed (x-helices and centrally placed repetitive β-turns.

RAPD analysis of aromatic and non-aromatic rice (Oryza sativa L.).

Genetic variation in nine aromatic and four nonaromatic rice varieties (Oryza sativa L.) was investigated at the DNA level using the randomly amplified polymorphic DNA (RAPD) technique. Twenty six random primers were used to amplify DNA segments and 177 PCR products were obtained of which 98 were polymorphic. One primer did not show polymorphism. A dendrogram showing the genetic distances of 13 rice varieties was constructed based on RAPD data.

Lathyrus Dehydrin : A Drought Inducible cDNA Clone — Isolation and Characterization

Dehydrin like transcripts (drought inducible mRNA) accumulated in drought stressed shoots and roots of Lathyrus sativus. ABA also induced the expression of dehydrin in unstressed seedlings but to a lower extent. A cDNA expression library was prepared in lambda ZAP vector from mRNA of drought stressed shoots of Lathyrus sativus cv P-24 and screened with pea dehydrin cDNA probe. One positive clone (∼ 500 bp) was purified and characterised. The partial nucleotide sequence of the clone Lsd, contained a possible open reading frame which would encode a polypeptide with region of significant amino acid sequence similarity to dehydrin from other plant species. The genomic counterpart of Lsd cDNA was found to be present within a fragment of ∼ 3.4 and ∼ 3.2 kb in parent as well as in the somaclones of Lathyrus sativus, by using different restriction enzymes.

Biochemical and Cytological Studies During Seed Development in Somaclones of Lathyrus sativus

Soluble proteins in six somaclones and the parent at three different stages of development of Lathyrus sativus seeds showed polypeptides of mol wts ranging from 20–165 kD on SDS-PAGE. Immature seeds showed eight distinct esterase bands and two peroxidase bands. Pattern for both the enzymes among somaclones was almost similar. During maturation a new isoform of peroxidase with high mobility appeared in all the somaclones, while one new isoform of intermediate mobility was visible in somaclones Bio L-08 and Bio I-08. Cytological analysis of all the somaclones confirmed the stability of the chromosome count of 2n=14. At metaphase I seven distinct bivalents and at anaphase I and II univalents showing complements of the seven chromosomes were visible. No structural or chromosomal aberration were observed among somaclones.

Developmental Expression of Microsomal Omega-6 Desaturase Gene (fad2-1) in Soybean Seeds

Fad2-1 gene encoded microsomal omega-6 desaturase plays a major role in controlling the conversion of oleic acid to linoleic acid within storage lipids during seed development. A 180 bp region (1241–1422) specific to fad2-1 was amplified and its sequence confirmed. Northern blot analysis using the labelled amplicon as probe indicated that the fad2-1 expression was induced during early stages of embryo development and peaked in the mid-maturation stages. Tissue specificity of fad2-1 transcripts was confirmed by the complete absence of fad2-1 transcripts in the vegetative tissues of leaf, stem and root. Developmental profile of fatty acids showed increased levels of linoleic acid during the mid-maturation stages which coincided with the enhanced expression of fad2-1 during the same period.

Genomic Organisation of α-Gliadin Gene in Wheat Varieties Differing in Chapati Characteristics

Wheat varieties, differing in chapati characteristics, showed marked restriction fragment length polymorphism when probed for gene encoding α-gliadin. EcoRI digested DNA from variety K-68 showed five hybridizing bands whereas Sonalika showed only three bands. BamHI and HindIII digested DNA from variety C-306 showed lesser number of hybridizing bands than Sonalika, while Pst I digested K-68 DNA showed six hybridizing bands. By screening of sub-genomic library of C-306, 10 clones encoding gliadin were isolated. Partial sequencing of a clone pBJ-1 (∼ 1.0 kb) showed polyglutamine coding region and an internal stop condon at 224 bp.

Stress Induced Polypeptides in Lathyrus sativus

Effect of stress during seed germination showed both qualitative and quantitative changes in respect to polypeptide pattern. In general, the number of polypeptides decreased with induced stress. Polypeptides of 46, 40, 35 and 28 kD were present even at sublethal dehydration state of the plant thereby indicating the possibility of their role in overcoming severe stress. This has also been confirmed by RNA dot-blot analysis using drought responsive DNA probe which showed an abundance of stress inducible transcripts on salinity, mannitol and ABA treatments. Salinity induced greater reduction in the expression of high molecular weight polypeptides than other stress treatments. ABA alongwith mannitol/salt induced higher synthesis of a 46 kD polypeptide.

Genomic Organization of α-Amylase Gene in High Lysine Opaque-2 Maize

Genomic DNA was isolated from 7-day old etiolated seedlings of normal and high lysine opaque-2 maize and purified by CsCl gradient. Purified DNA was found to be ∼48.5 kb in size. Restriction analysis of genomic DNA with EcoRI and HindIII did not reveal any noticeable difference between normal and opaque-2 DNA. Southern blot analysis, using α-amylase probe, showed multiple bands. One of the hybridizing bands (∼4 kb) of genomic DNA was more intense in opaque-2 than in normal. This DNA was cloned into pUC 18 plasmid and presence of α-amylase was confirmed by Southern hybridization using α-amylase probe.