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The symbiotic association of Piriformospora indica provides growth promotion leading to increased biomass as well as enhanced plant tolerance to biotic and abiotic stresses in a variety of plant species. Since various protein kinases are thought to be involved in mutual signaling in symbiotic interactions, we analyzed transcript profile of rice protein kinase genes during the infection process by real-time PCR. A rice L-type lectin protein kinase (OslecRK) and Arabidopsis AtLecRK genes are highly upregulated in roots during the initial stage of symbiotic association. In order to investigate the role of LecRK gene in root colonization, the Arabidopsis mutant line (Atlecrk) of rice homolog was used which shows the restricted intracellular penetration of the P. indica hyphae. The results indicate that LecRK might be a crucial factor in perception and recognition during plant–P. indica association by regulating fungal invasion.

LINE1 (L1) is an autonomous, non-LTR retrotransposon and the L1 family of retrotransposons constitute around 17%, 20% and 23% in the human, mouse and rat genomes respectively. Under normal physiological conditions, the retroelements remain by and large transcriptionally silent but are activated in response to biotic and abiotic stress conditions and during perturbation in cellular metabolism. They have also been shown to be transiently activated under certain developmental programs. Using RT-PCR, we show that the L1 elements are transcriptionally active in the hippocampus region of the brain of four-month-old rat under normal conditions without any apparent stress. Twenty non-redundant LINE1-specific reverse transcriptase (RTase) sequences form ORF2 region were isolated, cloned and sequenced. Full length L1 element sequences complementary to the isolated sequences were retrieved from the L1 database. In silico analysis was used to determine the presence of these retroelements proximal (up to 10 kb) to the genes transcriptionally active in the hippocampus. Many important genes were found to be in close proximity of the transcriptionally active L1 elements. Transcriptional activation of the elements possibly affects the expression of the neighbouring genes.

Increasing demand of fossil fuel and its depleting stock across the globe have necessitated a search for potential alternative sources of renewable energy. Recently focus has shifted to production of diesel from bio-oils derived from plant biomass as a potential source. However, it has been an endeavour to shift the exploration to non-edible oils produced by non-crop plants. Hiptage benghalensis is one such unexplored plant which stores ricinoleic acid in its seeds. In this report, analyses of various biochemical and biophysical parameters of H. benghalensis seed oil have been undertaken in order to explore the potential of this oil for biodiesel production. The plant was sourced from three different geographic regions of India. The total lipid and ricinoleic acid contents of seeds obtained from three locations varied from 54 to 69% and 69–87 %, respectively. Properties of various parameters of the H. benghalensis seed oil (HbSO), besides being non-edible prove its potential as a biodiesel feedstock. In order to test this potential, the seed oil was converted into biodiesel through the process of transesterification with an efficiency of ∼93 %. Analysis of various fuel properties like acid value, calorific value, viscosity, density, ash content, flash, pour and cloud point of the biodiesel in accordance with ASTM-D6751 standard and a comparison with the properties of other reported biodiesels showed its reasonable quality. Thus, this non-edible oil from H. benghalensis seeds has a potential to be utilized as an alternate green energy source for biodiesel production.

Pigeon pea (Cajanus cajan), an important legume crop is predominantly cultivated in tropical and subtropical regions of Asia and Africa. It is normally considered to have a low degree of genetic diversity, an impediment in undertaking crop improvement programmes. We have analysed genetic polymorphism of domesticated pigeon pea germplasm (47 accessions) across the world using earlier characterized panzee retrotransposon-based molecular markers. It was conjectured that since retrotransposons are interspersed throughout the genome, retroelements-based markers would be able to uncover polymorphism possibly inherent in the diversity of retroelement sequences. Two PCR-based techniques, sequence-specific amplified polymorphism (SSAP) and retrotransposon microsatellite amplified polymorphism (REMAP) were utilized for the analyses. We show that a considerable degree of polymorphism could be detected using these techniques. Three primer combinations in SSAP generated 297 amplified products across 47 accessions with an average of 99 amplicons per assay. Degree of polymorphism varied from 84–95%. In the REMAP assays, the number of amplicons was much less but up to 73% polymorphism could be detected. On the basis of similarity coefficients, dendrograms were constructed. The results demonstrate that the retrotransposon-based markers could serve as a better alternative for the assessment of genetic diversity in crops with apparent low genetic base.

Entamoeba histolytica contains a novel calcium-binding protein like calmodulin, which was discovered earlier, and we have reported the presence of its homologue(s) and a dependent protein kinase in plants. To understand the functions of these in plants, a cDNA encoding a calcium-binding protein isolated from Entamoeba histolytica (EhCaBP) was cloned into vector pBI121 in antisense orientation and transgenic tobacco plants were raised. These plants showed variation in several phenotypic characters, of which two distinct features, more greenness and leaf thickness, were inherited in subsequent generations. The increase in the level of total chlorophyll in different plants ranged from 60% to 70%. There was no major change in chloroplast structure and in the protein level of D1, D2, LHCP and RuBP carboxylase. These morphological changes were not seen in antisense calmodulin transgenic tobacco plants, nor was the calmodulin level altered in EhCaBP antisense plants.

Microbial communities in rhizosphere interact with each other and form a basis of a cumulative impact on plant growth. Rhizospheric microorganisms like Piriformospora indica and Azotobacter chroococcum are well known for their beneficial interaction with plants. These features make P. indica /A. chroococcum co-inoculation of crops most promising with respect to sustainable agriculture and to understanding the transitions in the evolution of rhizospheric microbiome. Here, we investigated interactions of P. indica with A. chroococcum in culture. Out of five Azotobacter strains tested, WR5 exhibited growth-promoting while strain M4 exerted growth-inhibitory effect on the fungus in axenic culture. Electron microscopy of co-culture indicated an intimate association of the bacterium with the fungus. 2-D gel electrophoresis followed by mass spectrometry of P. indica cellular proteins grown with or without WR5 and M4 showed differential expression of many metabolic proteins like enolase-I, ureaseD, the GTP binding protein YPT1 and the transmembrane protein RTM1. Fungal growth as influenced by bacterial crude metabolites was also monitored. Taken together, the results conform to a model where WR5 and M4 influence the overall growth and physiology of P. indica which may have a bearing on its symbiotic relationship with plants.

Deoxyribonuclease Activity from Acetone Powder Preparation of Chlamydomonas reinhardii

An immuno-homologue of maize Ca2+/calmodulin (CaM)-dependent protein kinase with a molecular mass of 72 kDa was identified in pea. The pea kinase (PsCCaMK) was upregulated in roots in response to low temperature and increased salinity. Exogenous Ca2+ application increased the kinase level and the response was faster than that obtained following stress application. Low temperature-mediated, but not salinity-mediated stress kinase increase was inhibited by the application of EGTA and W7, a CaM inhibitor. The purification of PsCCaMK using immuno-affinity chromatography resulted in coelution of the kinase with another polypeptide of molecular mass 40 kDa (p40). Western blot revealed the presence of PsCCaMK in nuclear protein extracts and was found to phosphorylate p40 in vitro. Gel mobility shift and South-Western analysis showed that p40 is a DNA-binding protein and it interacted specifically with one of the cis acting elements of the Arabidopsis CaM5 gene (AtCaM5) promoter. The binding of p40 to the specific elements in the AtCaM5 promoter was dependent of its dephosphorylated state. Our results suggest that p40 could be an upstream signal component of the stress responses.

By manipulating hormone levels, light intensities and temperature, we have developed an efficient leaf-disc method for the regeneration of plants via embryogenesis and for transformation in four genotypes of Vitis viniferaL. In MS basal medium supplemented with 1 mg l–16-benzylaminopurine (BAP) and 0.1 mg l–12,4-dichlorophenoxyacetic acid, leaf discs cultured for 2 weeks under dark conditions produced calli in over 80% of the cultures. These subsequently differentiated into pro-embryos and embryos only if kept under conditions of low light intensity (15 µE m–2 s–1) for 2 weeks before being transferred to conditions of high light intensity (60 µE m–2 s–1). If the calli were directly transferred to high light intensity, the differentiation into embryos was blocked and the calli turned pink. The somatic embryos germinated at a frequency of about 10% on NN basal medium and about 32% on NN medium supplemented with 1 mg l–1BAP and 0.1 mg l–1indole-3-butyric acid. The embryos, however, germinated when pre-exposed to a low temperature of 4°C for 2 weeks. If they were transferred directly to room temperature under conditions of high light intensity (60 µE m–2 s–1), shoot buds were produced, whereas under conditions of low light intensity (15 µE m–2 s–1) secondary embryogenesis was induced. About 90–95% of the in vitro grown plantlets could be successfully transferred to soil. The above method was also applicable for developing transgenic embryos whose transgenic nature was monitored using β-glucuronidase as a reporter gene.

The chapter describes human genome projects led independently by an international collaboration of countries and a private company. The human genome sequence has opened up a whole new chapter of understanding. How this information is revolutionizing the study of biology and medicine is elaborated upon.

This work was carried out to study the effects of vanadate on the expression of liver-type arginase in experimentally induced diabetes in the rat. The results showed that the activity and mRNA levels of arginase were increased significantly in the diabetic condition. Vanadate treatment reversed the increased activity and restored mRNA levels of arginase almost to the control values. The reversal effects of vanadate were found to be similar to those of insulin, and this further confirms the insulin-like effects of vanadate. ELISA and slot-blot assay observations were consistent with the biochemical measurements of enzyme activity. The increase in arginase activity and mRNA levels in diabetes and the decrease in insulin- and vanadate-treated animals may be due to the transcriptional regulation of arginase.

The high degree of conservation of nucleotide sequences among different members of a multigene family poses problems in analysis of expression patterns governed by each member of the gene family. In this report we describe a simple, semi-quantitative and single tube multiplex RT-PCR assay for simultaneous and relative expression analysis with an application to all the six members of Arabidopsis calmodulin multigene family. In the multiplex primer set, individual gene specific primers were derived from 3'-untranslated region of the genes and a single common primer from the conserved exonic region. Transcriptional activation of all the members of the calmodulin gene family in response to touch was monitored. The results demonstrate that two of the genes are not regulated by touch; however, the other four that are induced by touch show a differential response including their kinetics of induction.

We have cloned and characterized one of the genes that encode phenylalanine ammonia-lyase fromArabidopsis thaliana. A 1.8-kb fragment containing the upstream region of this gene,PALI, was transcriptionally fused to the s-glucuronidase reporter gene, and the construct was introduced into tobacco byAgrobacterium-mediated leaf disc transformation. The distribution of s-glucuronidase activity in the transgenic plants was analysed histochemically. The spatial pattern of activity showed that thePALI promoter is very active in the vascular tissues. No expression was observed in epidermal cells, trichomes and apical meristems. Activation of thePALI promoter in the early stages of seedling development remained confined to the regions adjacent to the root apical meristems; in later stages of seedling development, the pattern of expression was drastically altered. The results suggest that thePALI promoter is under the control of a complex set of signal transduction pathways which lead to its activation in response to tissue-specific, wounding and developmental cues.

The effect of the major flowering gene (PPD) on seed weight of chickpea was studied on 450 F3 families from reciprocal crosses between a small-seeded, early-flowering (PPD/PPD) type and a large-seeded, late flowering (PPD/PPD) cultivar. F4 progeny tests were carried out to determine the PPD genotypes of each individual F3. The effects of the PPD gene and the polygenes on mean seed weight were both significant. Genetic correlations between time to flowering and seed weight were positive and relatively high, suggesting that in certain genetic backgrounds it might be difficult to breed early-flowering cultivars without compromising seed weight.

Quorum Sensing (QS) is a natural phenomenon of communication circuits to regulate a diverse array of physiological activities. QS regulates microbial diversity and ecological balance through various process, i.e., symbiosis, motility, sporulation, virulence, biofilm formation, competence, conjugation, and antibiotic production. There are numerous bacteria, fungi, and plants that have components of a QS system to control a specific phenotype of an organism and to shape the microbial community in the rhizophere. Bacterial intercellular communication is based on the detection of diffusible signal molecules and their transduction mechanisms which convert the information into appropriate molecular–cellular responses. Recent advances in studies of QS systems have brought the understanding that biocommunication and community behavior are crucial parameters for the successful interactions in a mycorrhizosphere. We are only at the beginning to understand how plants control microbial communities in the rhizophere. Interspecies and cross-kingdom communication is implicated in successful symbiotic interactions of a variety of bacteria with fungi and plant systems.

We report a Ty3-gypsy like retrotransposon CARE1 (Cicer arietinum retro-element 1) in chickpea (Accession no. DQ239702). This 5,920-bp AT-rich (63%) element carries 723-bp 5'-LTR and 897-bp 3'-LTR flanking to an internal region of 4,300-bp. The LTRs of CARE1 show 93.9% nucleotide identity to each other and have 4-bp (ACTA) terminal inverted repeats. A 17-bp potential tRNA(met) primer binding site downstream to 5'-LTR and a 13-bp polypurine tract upstream to 3'-LTR have been identified. The order of characteristic domains (Gag-proteinase-reverse transcriptase-RNaseH-integrase) in the deduced amino acid sequence and its phylogenetic analysis with other retrotransposons, places CARE1 in the gypsy group of retrotransposons. Homologues of a number of cis-elements including CCAAT, TATA and GT-1 have been detected in the regulatory region or the 5'-LTR of CARE1. Transgenic tobacco plants containing 5'-LTR:GUS construct showed that its 5'-LTR is inactive in a heterologous system under normal as well as tissue culture conditions. Genomic Southern blot experiments using 5'-LTR of the element as a probe showed that CARE1 or its related elements are present in the genomes of various chickpea accessions from various geographic regions.

We report a novel Ty3-gypsy long terminal repeat retrotransposon CARE1 (_Cicer arietinum_ retro-element 1) in chickpea. This 5920-bp AT-rich (63%) element carries 723-bp 5' and 897-bp 3' LTRs respectively flanking an internal region of 4300-bp. The LTRs of CARE1 show 93.9% nucleotide identity to each other and have 4-bp (ACTA) terminal inverted repeats. A 17-bp potential tRNAmet primer binding site downstream to 5' LTR and a 13-bp polypurine tract upstream to 3' LTR have been identified. The order of domains (Gag-proteinase-reverse transcriptase-RNaseH-integrase) in the deduced amino acid sequence and phylogenetic tree constructed using reverse transcriptase sequences places CARE1 in the gypsy group of retrotransposons. Homologues of a number of _cis_-elements including CCAAT, TATA and GT-1 have been detected in the regulatory region or the 5' LTR of CARE1. Transgenic tobacco plants containing 5' LTR:GUS construct show that its 5'-LTR is inactive in a heterologous system under normal as well as tissue culture conditions. Genomic Southern blot experiments using 5’LTR of the element as a probe show that CARE1 or its related elements are present in the genomes of various chickpea accessions from various geographic regions.

We report a novel Ty3-gypsy long terminal repeat retrotransposon CARE1 (Cicer arietinum retro-element 1) in chickpea. This 5920-bp AT-rich (63%) element carries 723-bp 5' and 897-bp 3' LTRs respectively flanking an internal region of 4300-bp. The LTRs of CARE1 show 93.9% nucleotide identity to each other and have 4-bp (ACTA) terminal inverted repeats. A 17-bp potential tRNAmet primer binding site downstream to 5' LTR and a 13-bp polypurine tract upstream to 3' LTR have been identified. The order of domains (Gag-proteinase-reverse transcriptase-RNaseH-integrase) in the deduced amino acid sequence and phylogenetic tree constructed using reverse transcriptase sequences places CARE1 in the gypsy group of retrotransposons. Homologues of a number of cis-elements including CCAAT, TATA and GT-1 have been detected in the regulatory region or the 5' LTR of CARE1. Transgenic tobacco plants containing 5' LTR:GUS construct show that its 5'-LTR is inactive in a heterologous system under normal as well as tissue culture conditions. Genomic Southern blot experiments using 5’LTR of the element as a probe show that CARE1 or its related elements are present in the genomes of various chickpea accessions from various geographic regions.

Retrotransposons constitute a major fraction of plant genomes and these elements may have played a significant role in evolution and sequence organization of genomes. In order to access the diversity of Ty1-copia group of retroelements, reverse transcriptase (RT) sequences were amplified from chickpea genome, using the primers derived from two conserved domains of RT region. Thirty-six RT regions from independent amplicons were cloned and sequenced. On the basis of homology of deduced amino acids, the RT sequences could be grouped into three major families. The intra-family divergence at amino acid level ranges from 2 to 19%. Though intra-family RT sequences were conserved but no two sequences were identical. The results indicate a high degree of heterogeneity among the Ty1-copia group of retroelements from chickpea. It was possible to isolate RT specific sequences from RNA isolated from stressed seedlings, indicating that some of the retroelements may be functional under certain stress conditions.

An immuno-homologue of maize Ca2+/calmodulin (CaM)-dependent protein kinase with a molecular mass of 72 kDa was identified in pea. The pea kinase (PsCCaMK) was upregulated in roots in response to low temperature and increased salinity. Exogenous Ca2+ application increased the kinase level and the response was faster than that obtained following stress application. Low temperature-mediated, but not salinity-mediated stress kinase increase was inhibited by the application of EGTA and W7, a CaM inhibitor. The purification of PsCCaMK using immuno-affinity chromatography resulted in coelution of the kinase with another polypeptide of molecular mass 40 kDa (p40). Western blot revealed the presence of PsCCaMK in nuclear protein extracts and was found to phosphorylate p40 in vitro. Gel mobility shift and South-Western analysis showed that p40 is a DNA-binding protein and it interacted specifically with one of the cis acting elements of the Arabidopsis CaM5 gene (AtCaM5) promoter. The binding of p40 to the specific elements in the AtCaM5 promoter was dependent of its dephosphorylated state. Our results suggest that p40 could be an upstream signal component of the stress responses.

A fold back sequence linked to a copy of the transposable element, Tam2, has been Isolated, from a genomic library constructed using partially digested and size-fractionated Antirrhinum DNA Into λ EMBL4 vector, by using Tam2 specific probe. The fold back sequence visualized by heteroduplex analysis reveals a typical stem-loop structure. The foldback element is around 1.2 kb with an average of 227 bp of Inverted termini forming the stem.

At least eleven somatic compatibility loci exist in the myxomycete Didymium iridis. Cell fusion is controlled by at least seven fusion loci (Fusl-Fus7). Cytoplasmic compatibility is controlled by at least four clear-zone loci (Czl-Cz4). Plasmodia with identical phenotypes at all seven fusion loci, but different phenotypes at the clear-zone loci, will fuse temporarily, but fusion is soon blocked by cytoplasmic reactions which prevent complete mixing. Areas which contain cytoplasm from two incompatible plasmodia become clearly delineated from healthy cytoplasm. Such areas, termed clear zones, have been isolated and found to recover. If clear zones are sectioned into several small pieces, not all pieces will recover, indicating that toxic cytoplasmic reactions have occurred. Plasmodial fusion studies and F1 studies of recovered clear zones indicate that the clear-zone loci may also control nuclear survival in heterokaryons. CYTOPLASMIC INCOMPATIBILITY has been studied in the true slime molds (Clark and Collins, 1973a, b), in the higher fungi (Esser and Kuenen, 1967), and in Protozoa (Hildemann, 1972). The relatively small size of most protists makes it difficult to observe cytoplasmic interactions in these organisms. Slime mold plasmodia, however, are giant cells and cytoplasmic interactions upon cell fusion are easily observable at 45 x with a dissecting microscope. In addition, cytoplasmic interactions occur within 1-5 min after cell contact. Somatic cell compatibility in plasmodia of the true slime mold Didynium iridis (Ditmar) Fries is controlled by a system of at least eleven loci each with dominant and recessive alleles (Ling and Collins, 1970a, b; Ling, 1971; Collins and Ling, 1972). Two plasmodia must be phenotypically identical at all these somatic compatibility loci in order to fuse freely (Fig. 1). These loci can be categorized into two classes, depending on whether they allow a temporary fusion or no fusion at all (Ling and Ling, 1974). Fusion and heterokaryon production do not occur if two plasmodia differ phenotypically at any of the seven fusion loci Fusl-Fus7 (Fig. 2). Temporary fusion is controlled by four clearzone loci (Czl-Cz4). Fusion between two plasmodia with phenotypic differences at any of the clear-zone loci is terminated within a few minutes by coagulation reactions (Clark and Collins, 1 Received for publication 13 July 1973. Research supported by PHS Grant GM19165 and a University of Delaware Research Foundation Grant to the first author. The competent assistance of Andrew Liu is gratefullv acknowledged. 1973a, b; Upadhyaya and Ling, 1972; Ling and Upadhyaya, 1973). Areas formed by intermixing of two incompatible plasmodia lose pigment and appear quite different from the normal surrounding protoplasm (Fig. 3). These affected areas have been termed "clear zones" (Collins and Clark, 1968). Clear-zone formation has often been found to be directional. When plasmodia of two different phenotypes are repeatedly tested, most of the clear zones are formed in one member of the pair (Clark and Collins, 1973a; Ling and Upadhyaya, 1973). Clark and Collins (1973a) assumed that toxic reactions occurred within the clear zones. It was observed by previous workers that veins of living protoplasm often appeared within clear zones, but these were interpreted as invasion by healthy plasmodia. However, in the current study it was determined that if clear zones were excised from the surrounding plasmodia, recovery still took place and thus heterokaryons were produced. It is still quite possible that both invasion by healthy plasmodium and recovery normally take place within clear zones. This communication reports for the first time the reco,very pattern of heterokaryons formed between plasmodia differing at one or more clearzone loci. The fusion properties and survival of marker alleles in recovered clear zones are discussed in detail. MATERIALS AND METHODS-Plasmodia chosen for these studies were phenotypically identical at all fusion loci Fusl-Fus7. Thus fusions were possible, but extensive mixing of protoplasm was pre

Several alleles of the nivea locus of Antirrhinum majus, both stable and unstable, have been characterised genetically (Harrison and Carpenter 1973 a, b). In this work the niv-44 allele is characterised at the molecular level. It contains a 5kb insertion element, Tam 2, which has 14 base pair inverted repeats. There is a three base pair duplication at the target site, which is at the first intron-exon boundary of the chalcone synthase gene. Tam 2 homologous sequences are present in multiple copies in several A. majus lines, including niv-53, and most have at least a 2.9 kb sequence in common with the copy at the chalcone synthase gene. Possible reasons for the apparent stability of the niv-44 allele and molecular explanations for the role of this allele in paramutation in A. majus are discussed.

Summary Mitochondria and nuclei were isolated from excised germinating barley embryos and the incorporation of ( 3 H)-thymidine into the two organelle fractions was measured in order to determine the time of synthesis of mitochondrial DNA (mtDNA) in relation to the synthesis of nuclear DNA (nDNA). The results indicate that in barley seeds, nDNA synthesis begins at the 10th hour after soaking the excised embryos, and continues till 20th hour. The highest synthetic activity is between 13 and 17 hours. The mtDNA synthesis begins after the onset of the first S-phase but most of the mtDNA is synthesized late in the S-phase, between 15 and 17 hours after the initial soaking.