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Mohan B Singh

Publications and source records attributed to Mohan B Singh.

16 recordsLinked to original sources
PubMed2006

Histone H3 variants in male gametic cells of lily and H3 methylation in mature pollen.

Histones are vital structural proteins of chromatin that influence its dynamics and function. The tissue-specific expression of histone variants has been shown to regulate the expression of specific genes and genomic stability in animal systems. Here we report on the characterization of five histone H3 variants expressed in Lilium generative cell. The gcH3 and leH3 variants show unique sequence diversity by lacking a conserved lysine residue at position 9 (H3K9). The gH3 shares conserved structural features with centromeric H3 of Arabidopsis. The gH3 variant gene is strongly expressed in generative cells and gH3 histone is incorporated in to generative cell chromatin. The lysine residue of H3 at position 4 (H3K4) is highly methylated in the nuclei of generative cells of mature pollen, while methylation of H3K4 is low in vegetative cell nuclei. Taken together, these results suggest that male gametic cells of Lilium have unique chromatin state and histone H3 variants and their methylation might be involved in gene regulation of male gametic cells.

Amino Acid Sequence↗
PubMed2006

Transcriptional repression distinguishes somatic from germ cell lineages in a plant.

In flowering plants, the male germline begins with an asymmetric division, after which one of the resulting cells, the generative cell, divides symmetrically to produce two sperm cells. We show here that the male germline is initiated by transcriptional control. We identify GRSF, germline-restrictive silencing factor, from the lily. GRSF is ubiquitous in nongerm cells and is absent from male germ cells. GRSF recognizes silencer sequences in promoters of genes specific to the germline, stably repressing these genes in cells that are not destined to become germ cells.

Amino Acid Motifs↗
PubMed2006

Plant stem cells carve their own niche.

Stem cells are the precursors of differentiated cells and are, thus, indispensable for growth and development in plants and animals. Stem cells from both types of organisms share the fundamental features of a capacity for self-renewal and an ability to generate differentiated cells. The maintenance of stem cells in both systems is dependent upon reciprocal signalling between stem cells and the specialized tissue microenvironment known as the niche, which provides intercellular signals for stem cell regulation. One significantly underexplored facet of plant stem cells is the nature of their intrinsic transcriptional programme. A potentially rich avenue for addressing this deficiency is to combine laser-assisted microdissection and genome-wide transcriptional profiling to unravel the molecular road map controlling plant stem cells and their niches.

Animals↗
PubMed2006

Expressed sequence tag analysis of Lilium longiflorum generative cells.

The generative cell, the male gametic cell progenitor in flowering plants, undergoes mitotic division to produce two sperm cells. We have examined the gene expression profile of the Lilium longiflorum (lily) generative cell by sequencing expressed sequence tags (ESTs). A total of 886 ESTs derived from the generative cell cDNA library were clustered into 637 unique ESTs comprising 123 cluster ESTs and 514 singleton ESTs. Thirty-nine percent of non-redundant ESTs showing similarity to Arabidopsis genes with known function were thus assigned putative functions. Genes related to the ubiquitin system were abundant, suggesting the key role of ubiquitin-dependent proteolysis in gametogenesis. A total of 168 and 129 non-redundant lily generative cell ESTs showed significant similarity to maize sperm cell ESTs and Arabidopsis male gametophyte-specific transcripts, respectively. Fifty-five ESTs appeared to have significant similarities to both maize sperm cell ESTs and Arabidopsis male gametophyte-specific genes, indicating conservation of male gamete-expressed genes across different plant genera. Thus our data provide a handle to identify Arabidopsis gamete-expressed genes and to investigate their function. Several of these genes are potential candidates for analyzing the molecular basis of fertilization and for investigating mechanisms of gamete-specific transcriptional regulation in Arabidopsis through bioinformatics-based approaches.

Arabidopsis↗
PubMed2006

Recombinant expression systems for allergen vaccines.

Allergen immunotherapy of future is likely to be based on allergy vaccines that contain engineered allergens modified to abolish or substantially reduce their IgE-binding activity in order to remove the risk of unwanted anaphylactic responses. The development of efficient systems for the production of recombinant allergens in sufficient quantities is requirement for establishing use of engineered allergens as components of allergy vaccines. This review outlines relative advantages and disadvantages of various heterologous systems for production of recombinant allergens. Microbial systems are most convenient and cost effective platforms for the production of recombinant allergens. However, lack of post-translational processing implies that some allergens have to be expressed in eukaryotic systems for proper folding and post-translational modifications such as glycosylation. Yeast systems can yield high levels of recombinant allergens but often are associated with hyper- glycosylation problems. Mammalian cell culture systems offer suitable post -translational modifications but are nearly hundred fold more expensive than microbial systems. The use of plants as bio-factories for production of recombinant allergens is emerging as a very attractive option as plants-based production system offer several advantages over other expression systems such as post translational processing of proteins, low production costs, scale up ability and enhanced safety due to absence of animal or human pathogens.

Allergens↗
PubMed2005

Molecular control of stem cell maintenance in shoot apical meristem.

Sustained post-embryonic organ initiation and development in plants depends on coordinating the formation and differentiation of pluripotent stem cells in apical meristems. Transcriptional regulation and intercellular signalling appear to play key roles in this coordination process. Here we discuss the current knowledge about the molecular regulation of stem cell maintenance in the shoot apical meristem and recent attempts to delineate the molecular signatures of "stemness" in flowering plants. We also outline contemporary molecular approaches for deciphering the process of stem cell renewal in the shoot apical meristem.

Meristem↗
PubMed2005

Transcriptional activity of male gamete-specific histone gcH3 promoter in sperm cells of Lilium longiflorum.

Histones are essential for packaging of eukaryotic genomic DNA in nucleosomes, and histone gene expression is normally coupled with DNA synthesis. Some of the flowering plant histone genes show strictly male gamete-specific expression. However, mechanisms underlying their male gamete-specific expression have not been elucidated so far. Here we report the isolation of the male gamete-specific histone gcH3 promoter from Lilium longiflorum and its activity in the male gametic cell of the flowering plant. The OCT motif, which is well conserved in plant histone promoters regulating S phase-specific expression, is not conserved in the gcH3 promoter. Instead sequence motifs identical to GC box 1 and GC box 2, the transcriptional activator and suppressor for mammalian testis-specific histone H1t, are present in the gcH3 promoter, suggesting that plants and animals share the mechanism which governs the specificity of gene expression in male gametic cells. Male gamete-specific activation of the gcH3 promoter has been confirmed by microprojectile bombardment in lily pollen. The sperm cell carrying gold particles showed reporter gene expression, while green fluorescent protein (GFP) was absent in the other sperm cell which had no particles, confirming that the gcH3 promoter is activated in the male gametic cell, and sperm cells have transcriptional and translational machinery that is independent of the vegetative cell of pollen.

Animals↗
PubMed2005

Analysis of the histone H3 gene family in Arabidopsis and identification of the male-gamete-specific variant AtMGH3.

Histones are major components of chromatin, the protein-DNA complex involved in DNA packaging and transcriptional regulation. Histone genes have been extensively investigated at the genome level in animal systems and have been classified as replication dependent, replication independent or tissue specific. However, no such study is available in a plant system. In this paper we report that there are 15 histone H3 genes in the Arabidopsis genome, including five H3.1 genes, three H3.3 genes and five H3.3-like genes. A gene structure analysis revealed that gene duplication causes redundancy of the histone H3 genes. The expression of one of the H3 genes, termed AtMGH3/At1g19890, is cell-specific, being restricted to the generative and sperm cells of Arabidopsis pollen as shown by in situ hybridisation and reporter gene analysis. Thus, we conclude that in Arabidopsis, AtMGH3 is a male-gamete-specific histone H3 gene. A T-DNA insertion line for AtMGH3 revealed decreased expression and ectopic RNA splicing. The T-DNA insertion lines for AtMGH3/At1g19890 and other H3 genes revealed a normal growth phenotype and reproductive fertility. These findings suggest that other H3 genes are likely to compensate for the T-DNA-insertion-induced loss of a single H3 gene because of the high redundancy of these genes in the Arabidopsis genome. These T-DNA mutant lines should be useful for accumulating different H3 gene mutations in a single plant and for studying replication-dependent and replication-independent H3 genes and the specific role of AtMGH3 in chromatin remodelling and transcriptional regulation during development of male gametes.

Amino Acid Sequence↗
PubMed2004

Molecular characterization of polygalacturonases as grass pollen-specific marker allergens: expulsion from pollen via submicronic respirable particles.

Grass pollen belong to the most important allergen sources involved in the elicitation of allergic asthma. We have isolated cDNAs coding for Bermuda grass (Cynodon dactylon) and timothy grass (Phleum pratense) pollen allergens, belonging to a family of pectin-degrading enzymes (i.e., polygalacturonases). The corresponding allergens, termed Cyn d 13 and Phl p 13, represent glycoproteins of approximately 42 kDa and isoelectric points of 7.5. rPhl p 13 was expressed in Escherichia coli and purified to homogeneity. Immunogold electron microscopy using rabbit anti-rPhl p 13 Abs demonstrated that in dry pollen group 13, allergens represent primarily intracellular proteins, whereas exposure of pollen to rainwater caused a massive release of cytoplasmic material containing submicronic particles of respirable size, which were coated with group 13 allergens. The latter may explain respiratory sensitization to group 13 allergens and represents a possible pathomechanism in the induction of asthma attacks after heavy rainfalls. rPhl p 13 was recognized by 36% of grass pollen allergic patients, showed IgE binding capacity comparable to natural Phl p 13, and induced specific and dose-dependent basophil histamine release. Epitope mapping studies localized major IgE epitopes to the C terminus of the molecule outside the highly conserved functional polygalacturonase domains. The latter result explains why rPhl p 13 contains grass pollen-specific IgE epitopes and may be used to diagnose genuine sensitization to grass pollen. Our finding that rabbit anti-rPhl p 13 Abs blocked patients' IgE binding to the allergen suggests that rPhl p 13 may be used for immunotherapy of sensitized patients.

Allergens↗
PubMed2004

Knocking out expression of plant allergen genes.

Pollen of many grasses, trees, and weeds are the source of inhalant allergic proteins while various other plant products are allergenic only upon their ingestion as a food source. Allergenic proteins of pollen are exposed to human immune system after their rapid release from pollen upon coming in contact with moist surface of nasal mucosa. The advent of molecular cloning and ability to genetically transform plants now offer unprecedented opportunities to produce hypoallergenic plants by targeted switching off allergen production. Gene silencing strategies that operate at post-transcriptional level are highly suitable for blocking allergen production. We have demonstrated the concept of allergen gene silencing through antisense approach by producing ryegrass plants that do not produce major allergen in its pollen. Our results show the potential of antisense approach in reducing the allergenic potential of plants. Such a strategy can have a general applicability for production of transgenic plants depleted of both inhaled and ingested allergens. In addition, such an approach could also help in elucidating the in vivo function of allergen(s) in plants and contribution of an allergen to overall allergenic potential of an allergen source.

Allergens↗
PubMed2004

Engineered allergens for immunotherapy.

PURPOSE OF REVIEW: Specific immunotherapy is a clinically effective causative treatment for allergic conditions. However, the reagents used for immunotherapy are crude extracts prepared from natural sources with potential life-threatening anaphylactic side effects. Molecular cloning of allergens has made it feasible to design novel therapeutic approaches for improved and safer forms of allergen-specific immunotherapy. The purpose of this review is to examine recent advances made in the last 2 years in genetic engineering of allergens for specific immunotherapy. RECENT FINDINGS: Genetic engineering of allergen with nil or low IgE reactivity but retained T-cell reactivity offers a novel therapeutic approach to improving safety and efficacy of allergen-specific immunotherapy. Hypoallergenic forms of major allergens have been produced, with reduced IgE epitopes while preserving other characteristics of the molecule to induce a protective response. SUMMARY: Hypoallergenic forms of major allergens are potential candidates for allergen-specific immunotherapy in the future. These genetically engineered hypoallergens now need to be tested in clinical trials before being widely used. Safer and more efficacious vaccines would increase patient compliance leading to extensive use of immunotherapy.

Air Pollutants↗
PubMed2003

Isolation and characterization of a flowering plant male gametic cell-specific promoter.

Flowering plant male gametic cell-specific gene expression has been reported recently but the regulatory elements controlling specificity of such genes expressed in generative cell and sperm cells have not been identified and studied. Here, we report the 0.8 kb promoter sequence upstream of the start of the transcription site of the generative cell-specific gene, LGC1, sufficient to regulate the expression of reporter genes in a cell-specific manner. In addition, the diphtheria toxin A-chain- (DT-A)-coding region under the control of the LGC1 promoter sequence confirmed unequivocally the lack of LGC1 expression in vegetative tissues. Transgenic tobacco plants carrying the LGC1-DT/A construct showed normal phenotype except for anthers of these plants that contained sterile and aborted pollen. Truncation and internal deletion analysis of the LGC1 promoter identified -242 bp as the minimal sequence necessary for male gametic cell-specific expression. In addition, a regulatory sequence required for determining generative cell-specific expression of LGC1 was identified. Deletion of this regulatory sequence led to loss of the generative cell specificity resulting in activation of this promoter in other tissues where it is normally repressed. Therefore, male gametic cell specificity of the LGC1 gene seems to be regulated by factors that suppress its activation in other plant cells. This is the first report of a male gametic cell-specific promoter, hence can be used as a novel tool in molecular analyses and experimental manipulation of flowering plant spermatogenesis and fertilization.

Base Sequence↗
PubMed2003

Hypoallergenic derivatives of major grass pollen allergens for allergy vaccination.

Grass pollen-induced hay-fever and allergic asthma represent a major health problem in industrialized countries. Whereas the symptoms of these allergic conditions can be controlled by pharmacotherapy, specific immunotherapy vaccination is the only causative approach towards the treatment of these type 1 allergies. Specific immunotherapy is based on administration of increasing amounts of the disease-causing allergens in the form of allergen-containing extracts. However, the extracts used for immunotherapy consist of allergenic and non-allergenic components and may induce severe anaphylactic side-effects upon therapeutic administration. With recent developments in molecular biology of pollen allergens it has become feasible to produce modified hypoallergenic derivatives of recombinant allergens with abrogated or greatly reduced likelihood of anaphylactic side-effects as compared to extract-based treatments. We have demonstrated this concept through reducing the anaphylactic potential of major rye grass pollen allergens by introducing a few point mutations which leave the overall structural fold of the molecule unaltered. These modified forms are expected to make allergen-specific immunotherapy more widely used in the future.

Allergens↗
PubMed2003

Oral immunization with a recombinant major grass pollen allergen induces blocking antibodies in mice.

BACKGROUND: Immunotherapy for the treatment of pollen allergies traditionally involves a series of parenteral injections of a crude pollen extract. Successful application of this treatment results in the development of systemic tolerance to the sensitizing allergens, including the induction of blocking antibodies. OBJECTIVE: We sought to investigate whether oral immunization with a recombinant pollen allergen could induce a systemic immune response, and the production of systemic blocking antibodies in mice. METHODS: C57BL/10 mice were orally administered rLol p 5 or sodium chloride solution via gavage. RESULTS: We report that the oral administration of rLol p 5 induced a systemic immune response, including the induction of both blocking and interspecific cross-reactive antibodies. CONCLUSION: Our results suggest that oral administration of a major grass pollen allergen can induce the development of a systemic immune response including the production of systemic blocking and cross-reactive antibodies, a response that may offer immunological protection upon subsequent allergen exposure.

Administration, Oral↗
PubMed2002

Mutants of the major ryegrass pollen allergen, Lol p 5, with reduced IgE-binding capacity: candidates for grass pollen-specific immunotherapy.

More than 400 million individuals are sensitized to grass pollen allergens. Group 5 allergens represent the most potent grass pollen allergens recognized by more than 80 % of grass pollen allergic patients. The aim of our study was to reduce the allergenic activity of group 5 allergens for specific immunotherapy of grass pollen allergy. Based on B- and T-cell epitope mapping studies and on sequence comparison of group 5 allergens from different grasses, point mutations were introduced by site-directed mutagenesis in highly conserved sequence domains of Lol p 5, the group 5 allergen from ryegrass. We obtained Lol p 5 mutants with low IgE-binding capacity and reduced allergenic activity as determined by basophil histamine release and by skin prick testing in allergic patients. Circular dichroism analysis showed that these mutants exhibited an overall structural fold similar to the recombinant Lol p 5 wild-type allergen. In addition, Lol p 5 mutants retained the ability to induce proliferation of group 5 allergen-specific T cell lines and clones. Our results demonstrate that a few point mutations in the Lol p 5 sequence yield mutants with reduced allergenic activity that represent potential vaccine candidates for immunotherapy of grass pollen allergy.

Allergens↗