Irfan Ahmad Ghazi

The sequence of rice chromosomes 11 and 12, rich in disease resistance genes and recent gene duplications

Rice is an important staple food and, with the smallest cereal genome, serves as a reference species for studies on the evolution of cereals and other grasses. Therefore, decoding its entire genome will be a prerequisite for applied and basic research on this species and all other cereals. We have determined and analyzed the complete sequences of two of its chromosomes, 11 and 12, which total 55.9 Mb (14.3% of the entire genome length), based on a set of overlapping clones. A total of 5,993 non-transposable element related genes are present on these chromosomes. Among them are 289 disease resistance-like and 28 defense-response genes, a higher proportion of these categories than on any other rice chromosome. A three-Mb segment on both chromosomes resulted from a duplication 7.7 million years ago (mya), the most recent large-scale duplication in the rice genome. Paralogous gene copies within this segmental duplication can be aligned with genomic assemblies from sorghum and maize. Although these gene copies are preserved on both chromosomes, their expression patterns have diverged. When the gene order of rice chromosomes 11 and 12 was compared to wheat gene loci, significant synteny between these orthologous regions was detected, illustrating the presence of conserved genes alternating with recently evolved genes. Because the resistance and defense response genes, enriched on these chromosomes relative to the whole genome, also occur in clusters, they provide a preferred target for breeding durable disease resistance in rice and the isolation of their allelic variants. The recent duplication of a large chromosomal segment coupled with the high density of disease resistance gene clusters makes this the most recently evolved part of the rice genome. Based on syntenic alignments of these chromosomes, rice chromosome 11 and 12 do not appear to have resulted from a single whole-genome duplication event as previously suggested.BackgroundRice is an important staple food and, with the smallest cereal genome, serves as a reference species for studies on the evolution of cereals and other grasses. Therefore, decoding its entire genome will be a prerequisite for applied and basic research on this species and all other cereals.ResultsWe have determined and analyzed the complete sequences of two of its chromosomes, 11 and 12, which total 55.9 Mb (14.3% of the entire genome length), based on a set of overlapping clones. A total of 5,993 non-transposable element related genes are present on these chromosomes. Among them are 289 disease resistance-like and 28 defense-response genes, a higher proportion of these categories than on any other rice chromosome. A three-Mb segment on both chromosomes resulted from a duplication 7.7 million years ago (mya), the most recent large-scale duplication in the rice genome. Paralogous gene copies within this segmental duplication can be aligned with genomic assemblies from sorghum and maize. Although these gene copies are preserved on both chromosomes, their expression patterns have diverged. When the gene order of rice chromosomes 11 and 12 was compared to wheat gene loci, significant synteny between these orthologous regions was detected, illustrating the presence of conserved genes alternating with recently evolved genes.ConclusionBecause the resistance and defense response genes, enriched on these chromosomes relative to the whole genome, also occur in clusters, they provide a preferred target for breeding durable disease resistance in rice and the isolation of their allelic variants. The recent duplication of a large chromosomal segment coupled with the high density of disease resistance gene clusters makes this the most recently evolved part of the rice genome. Based on syntenic alignments of these chromosomes, rice chromosome 11 and 12 do not appear to have resulted from a single whole-genome duplication event as previously suggested.

Evaluation of Abelmoschus moschatus extracts for antioxidant, free radical scavenging, antimicrobial and antiproliferative activities using in vitro assays

BackgroundAbelmoschus moschatus Medik. leaves and seeds are considered as valuable traditional medicine. The aromatic seeds of this plant are aphrodisiac, ophthalmic, cardio tonic, antispasmodic and used in the treatment of intestinal complaints and check queasiness. To give a scientific basis for traditional usage of this medicinal plant, the seed and leaf extracts were evaluated for their antioxidant, free radical scavenging, antimicrobial and antiproliferative activities.MethodsIn this study, antioxidant, antimicrobial and antiproliferative activities of A. moschatus extracts were evaluated in a series of in vitro assay involving free radicals, reactive oxygen species and their IC50 values were also determined. The antioxidant activities of the seed and leaf extracts of A. moschatus were determined by total antioxidant, DPPH, and ferrous reducing antioxidant property (FRAP) methods. In addition, the antiproliferative activity was also evaluated using colorectal adenocarcinoma and retinoblastoma human cancer cell lines. Moreover, six bacterial reference strains, two gram-positive (Bacillus subtilis and Staphylococcus aureus), four gram-negative (Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris and Salmonella enterica paratyphi) and one fungal strain (Candida albicans) were used to evaluate its antimicrobial activity.ResultsThe results from this study showed that the antioxidant activities of A. moschatus as determined by the total phenol, flavonoids, total antioxidant and FRAP methods were higher in leaf than that of the seed extracts. On the other hand, the aqueous overnight seed extract (AMS-I) has shown significant radical scavenging activity as in 1, 1- Diphenyl-2-picrylhydrazyl (DPPH), hydrogen peroxide, hydroxyl radical, superoxide and lipid peroxidation as compared to other seed and leaf extracts. The AMS-I and AML-IV have shown activity against six and seven microorganisms respectively. Simulteneously, AMS-IV and AML-IV have demonstrated potential antiproliferative activity against two human cell lines - Colorectal adenocarcinoma (COLO-205) and retinoblastoma (Y79).ConclusionThe seed and leaf extracts of A. moschatus possess significant antioxidant activity and could serve as free radical inhibitors or scavenger, or substitute, probably as primary antioxidants. The plant possesses moderate antibacterial activity against bacterial strains used in this study. Hydroalcoholic seed and leaf extracts also exhibited antiproliferative activity against two human cancer cell lines. A. moschatus may therefore, be a good candidate for functional foods as well as pharmaceutics.

Single-copy genes define a conserved order between rice and wheat for understanding differences caused by duplication, deletion, and transposition of genes

The high-quality rice genome sequence is serving as a reference for comparative genome analysis in crop plants, especially cereals. However, early comparisons with bread wheat showed complex patterns of conserved synteny (gene content) and colinearity (gene order). Here, we show the presence of ancient duplicated segments in the progenitor of wheat, which were first identified in the rice genome. We also show that single-copy (SC) rice genes, those representing unique matches with wheat expressed sequence tag (EST) unigene contigs in the whole rice genome, show more than twice the proportion of genes mapping to syntenic wheat chromosome as compared to the multicopy (MC) or duplicated rice genes. While 58.7% of the 1,244 mapped SC rice genes were located in single syntenic wheat chromosome groups, the remaining 41.3% were distributed randomly to the other six non-syntenic wheat groups. This could only be explained by a background dispersal of genes in the genome through transposition or other unknown mechanism. The breakdown of rice–wheat synteny due to such transpositions was much greater near the wheat centromeres. Furthermore, the SC rice genes revealed a conserved primordial gene order that gives clues to the origin of rice and wheat chromosomes from a common ancestor through polyploidy, aneuploidy, centromeric fusions, and translocations. Apart from the bin-mapped wheat EST contigs, we also compared 56,298 predicted rice genes with 39,813 wheat EST contigs assembled from 409,765 EST sequences and identified 7,241 SC rice gene homologs of wheat. Based on the conserved colinearity of 1,063 mapped SC rice genes across the bins of individual wheat chromosomes, we predicted the wheat bin location of 6,178 unmapped SC rice gene homologs and validated the location of 213 of these in the telomeric bins of 21 wheat chromosomes with 35.4% initial success. This opens up the possibility of directed mapping of a large number of conserved SC rice gene homologs in wheat. Overall, only 46.4% of these SC genes code for proteins with known functional domains; the remaining 53.6% have unknown function, and hence, represent an important, but yet, under explored category of genes.

Cross-transferability and Polymorphic Potential of Genomic STMS Markers of Brassica Species

The present study was carried out with the objective of evaluating genomic STMS markers developed earlier in Brassica napus, B. oleracea, B. rapa and B. nigra for their use in Brassica juncea and B. carinata. Ninety-six of the 100 STMS markers used under standardized annealing temperatures and gel concentrations produced clear reproducible amplification pattern. For majority of the markers 60 °C annealing temperature and 3.5% metaphor agarose gel were found suitable. High cross-transferability of STMS markers to related Brassica species including B. carinata (91.6%) and B. juncea (87.5%) suggested the possibility of utilizing these markers for genome analysis in the species where no such markers are available. The ‘B’ genome derived markers showed lower level of transferability to the ‘A’ and ‘C’ genome Brassica species. The potential of STMS markers to detect polymorphism among Brassica species and genera was 98.9%. The level of inter-specific polymorphism was much higher than the intea-specific polymorphism. The markers capable of revealing polymorphism among Brassica species and genera would be useful in Brassica introgression breeding programme. The polymorphic markers were found efficient in establishing the expected evolutionary relationships among the six different Brassica species and two related genera. Low level of intra-specific polymorphism revealed by these markers suggested use of a large set of such markers for various applications in Brassica genetics, genomics and breeding.

Physical mapping, expression analysis and polymorphism survey of resistance gene analogues on chromosome 11 of rice

Rice is the first cereal genome with a finished sequence and a model crop that has important syntenic relationships with other cereal species. The objectives of our study were to identify resistance gene analogue (RGA) sequences from chromosome 11 of rice, understand their expression in other cereals and dicots by in silico analysis, determine their presence on other rice chromosomes, and evaluate the extent of polymorphism and actual expression in a set of rice genotypes. A total of 195 RGAs were predicted and physically localised. Of these, 91.79% expressed in rice, and 51.28% expressed in wheat, which was the highest among other cereals. Among monocots, sugarcane showed the highest (78.92%) expression, while among dicots, RGAs were maximally expressed in Arabidopsis (11.79%). Interestingly, two of the chromosome 11-specific RGAs were found to be expressing in all the organisms studied. Eighty RGAs of chromosome 11 had significant homology with chromosome 12, which was the maximum among all the rice chromosomes. Thirty-one per cent of the RGAs used in polymerase chain reaction (PCR) amplification showed polymorphism in a set of rice genotypes. Actual gene expression analysis revealed post-inoculation induction of one RGA in the rice line IRBB-4 carrying the bacterial blight resistance gene Xa-4. Our results have implications for the development of sequence-based markers and functional validation of specific RGAs in rice.

Bioassay-Guided Fractionation and In Vitro Antiproliferative Effects of Fractions of Artemisia nilagirica on THP-1 cell line

ABSTACT Artemisia nilagirica (Clarke) is a widely used medicinal herb in Indian traditional system of medicine. Therefore, the present study was designed to evaluate the effects of A. nilagirica extracts/fractions on inhibition of proliferation and apoptosis in a human monocytic leukemia (THP-1) cell line. The crude extracts (A. nilagirica ethyl acetate extract [ANE] and A. nilagirica methanolic extract [ANA]) showed cytotoxic activity toward THP-1 cells with the IC50 values of 38.21 ± 7.37 and 132.41 ± 7.19 µg/ml, respectively. However, the cytotoxic activity of active fractions (ANE-B and ANM-9) obtained after column chromatography was found to be much more pronounced than their parent extracts. The IC50 values of ANE-B and ANM-9 were found to be 27.04 ± 2.54 µg/ml and 12.70 ± 4.79 µg/ml, respectively, suggesting greater susceptibility of the malignant cells. Cell cycle analysis and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay revealed that inhibition of cell growth by A. nilagirica fractions on THP-1 cells was mediated by apoptosis. Active fractions of A. nilagirica increased the expression levels of caspase-3, −7, and poly-ADP-ribose polymerase (PARP), a critical member of the apoptotic pathway. These results suggested that active fractions of A. nilagirica may play a promising role in growth suppression by inducing apoptosis in human monocytic leukemic cells via mitochondria-dependent and death receptor-dependent apoptotic pathways.

Nucleotide diversity analysis of three major bacterial blight resistance genes in rice.

Nucleotide sequence polymorphisms among R gene alleles influence the process of co-evolutionary interaction between host and pathogen by shaping the response of host plants towards invading pathogens. Here, we present the DNA sequence polymorphisms and diversities present among natural alleles of three rice bacterial blight resistance genes, Xa21, Xa26 and xa5. The diversity was examined across different wild relatives and cultivars of Oryza species. Functional significance of selected alleles was evaluated through semi-quantitative reverse transcription polymerase chain reaction and real time PCR. The greatest nucleotide diversity and singleton variable sites (SVS) were present in Xa26 (π = 0.01958; SVS = 182) followed by xa5 and Xa21 alleles. The highest frequency of single nucleotide polymorphisms were observed in Xa21 alleles and least in xa5. Transition bias was observed in all the genes and ‘G’ to ‘A’ transitions were more favored than other form of transitions. Neutrality tests failed to show the presence of selection at these loci, though negative Tajima’s D values indicate the presence of a rare form of polymorphisms. At the interspecies level, O. nivara exhibited more diversity than O. sativa. We have also identified two nearly identical resistant alleles of xa5 and two sequentially identical alleles of Xa21. The alleles of xa5 showed basal levels of expression while Xa21 alleles were functionally not expressed.

Cytoprotective Activity and Anti-inflammatory Properties of Artemisia nilagirica (Clarke) Extracts-A Study with Macrophages

Abstract Macrophages are prone to oxidative stress which can result either in severe damage to their function or even cell death. The present study was aimed to reveal the cytoprotective potential of Artemisia nilagirica (clarke.) extracts against tertiary butyl hydroperoxide (t-BHP) induced cell death and possible anti-inflammatory potential using RAW 264.7 murine macrophages. The extracts were shown to provide cytoprotective action in t-BHP treated cells via inhibition of lipid peroxidation, ROS generation and restoration of mitochondrial membrane potential. There was a significant change in the expression of genes for antioxidant enzymes in the cells which helped the cells to ameliorate the damaged biochemical machinery within cells. Furthermore, the extracts displayed a prominent downregulation of inducible nitric oxide synthase (iNOS) which led to a reduction of nitric oxide in lipopolysaccharide-treated RAW 264.7 cells that indicated the anti-inflammatory potential of these extracts. It was also found that at least by two of the extracts the iNOS downregulation is mediated by induction of Hemoxygenase-1. The GC-MS evaluation of extracts was carried out to identify the phytochemical constituents. The overall results specified that A. nilagirica can be a potential source for protective agents against oxidative stress and act as a reservoir of anti-inflammatory compounds.

Growth Inhibition and Apoptosis Inducing Effects of Artemisia absinthium L. Fractions on Chronic Myeloid Leukemia (K562) Cells

Abstract Plant-derived active principles may serve as potent and cost effective alternative medicinal strategy for human ailments. Artemisia absinthium is a potent medicinal plant growing wild in Kashmir Himalayan region and used traditionally for several ailments. The objective of present study was to evaluate the cytotoxic effects of Artemisia absinthium extracts on Chronic myeloid leukemia cells (K562) followed by bioassay-guided fractionation and evaluation of cell death mechanism. Two potent fractions (AAH-7) and (AAM-5) were obtained which inhibited the cells exhibiting IC50 values of 20.95 ± 2.60 μg/ml and 15.30 ± 1.24 μg/ml respectively and were comparatively less lethal to normal cells. The fractions led to chromatin condensation and DNA fragmentation in cells suggesting apoptosis. The fractions also led to an enhancement of intracellular reactive oxygen species (ROS) and diminution of mitochondrial membrane potential (MMP) leading to apoptosis. Flow cytometric analysis revealed the modulation of the cell cycle pattern and phosphatidylserine externalization. Treatment with fractions led to cleavage of Caspase-3, Poly ADP-ribose polymerase (PARP), decrease in the expression of Bcl-2 protein (anti-apoptotic) and an increase in expression of Bax protein (pro-apoptotic). The main constituents detected by GC-MS and LC-ESI-MS mainly included terpenoids which are fragrance producing components of Artemisia species.

Phytochemical content and antioxidant potential of Clerodendrum inerme and its different parts-A comparative study

Abstract Medicinal plants are excellent sources for phytochemicals, mostly polyphenols, flavonoids which exhibit high antioxidant activity. The therapeutic efficacy of these antioxidants for specific pharmacological actions has also been established by experimental and clinical studies. The aim of this study was to investigate the phytochemical and antioxidant properties of different parts of Clerodendrum inerme in a series of in vitro assays in order to find possible sources of antioxidants in food and pharmaceutical formulations. The whole plant of C. inerme and its different parts were extracted by cold and hot extraction methods with 95% ethanol and subsequently evaluated for phytochemical and in vitro antioxidant screening. The total antioxidant capacity of leaf extracts was found to be 10.41±0.321 mg AAE/g dw and the ferric reducing power was found to be 45.39±3.563 mg AAE/g dw. The IC50 values for free radicals scavenging were found to be lowest in case of leaf extracts compared to other extracts; for DPPH (30.93±0.6569 μg/ml), H2O2 (37.37±1.107 μg/ml), Super oxide (44.10±1.177 μg/ml) and lipid peroxidation (29.32±1.106 μg/ml). The data from present results revealed that C. inerme act as an antioxidant agent due to its free radical scavenging activity and can be utilised as an effective and safe source of functional food materials such as natural antioxidants.