Evolution, function, and regulation of genomic imprinting in plant seed development

Genomic imprinting is an epigenetic phenomenon whereby genetically identical alleles are differentially expressed dependent on their parent-of-origin. Genomic imprinting has independently evolved in flowering plants and mammals. In both organism classes, imprinting occurs in embryo-nourishing tissues, the placenta and the endosperm, respectively, and it has been proposed that imprinted genes regulate the transfer of nutrients to the developing progeny. Many imprinted genes are located in the vicinity of DNA-methylated transposon or repeat sequences, implying that transposon insertions are associated with the evolution of imprinted loci. The antagonistic action of DNA methylation and Polycomb group-mediated histone methylation seems important for the regulation of many imprinted plant genes, whereby the position of such epigenetic modifications can determine whether a gene will be mainly expressed from either the maternally or paternally inherited alleles. Furthermore, long non-coding RNAs seem to play an as yet underappreciated role for the regulation of imprinted plant genes. Imprinted expression of a number of genes is conserved between monocots and dicots, suggesting that long-term selection can maintain imprinted expression at some loci.     

Glycoproteomic survey of Mammillaria gracillis tissues grown in vitro

The structure elucidation of protein-linked N-glycans in plants has raised interest in the past years due to remarkable physiological roles attributed to these modifications. However, little information about the glycoprotein patterns related to plant cell differentiation, dedifferentiation and transformation is available. In this work, the use of two-dimensional polyacrylamide gel electrophoresis in conjunction with matrix assisted laser/desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) for the characterization of carbohydrates released from plant glycoproteins is described. Proteins from different Mammillaria tissues (shoot, callus, hyperhydric regenerant, and TW tumor) were separated by 2D SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane and incubated with Con A to detect N-glycosylated proteins. To discover if the same protein can have various N-glycan structures depending on the organization status of the tissue, the selected glycoprotein spot, which was common for all investigated tissues, was excised from the gels and digested by PNGase A. The released oligosaccharides were analyzed by MALDI-TOF-MS. The results obtained in this study indicate that the N-glycosylation pattern of the protein is clearly dependent on level of plant tissue organization and can be related to the specific morphogenic status.     

Immunoprofiling of pectic polysaccharides

An assay is described for the rapid identification of unbranched homogalacturonan and branched components occurring in samples of pectic polysaccharides using anti-pectin monoclonal antibodies. The assay involves the immunodetection of pectic polysaccharides after separation into two components during the application in small volumes to nitrocellulose. In this system, homoglacturonan-rich components migrate further on the nitrocellulose in contrast to pectic components with abundant side chains, resulting in a clear separation and discrete rings of distinct polysaccharides that can be identified using specific antibodies. This procedure is also directly applicable to preparations of plant material without the need for isolation of pectic polysaccharides.     

Immunogold for detection of antigen on nitrocellulose paper

The application of immunogold for the detection of antigen-antibody complexes on nitrocellulose paper is described. Tobacco mosaic virus (TMV) protein, either purified or in leaf extract, was bound to the nitrocellulose paper and then exposed to rabbit anti-TMV serum. The antigen-antibody complexes were detected by gold-labeled goat anti-rabbit immunoglobulin G. The gold-labeled antibody is directly visible because of its pink color. This method can detect 1-5 pg of TMV protein, either in purified form or in the unpurified plant extract, with high specificity.     

Silver Impregnation of the Golgi Apparatus, with Subsequent Nitrocellulose Embedding

The appearance of silver impregnation of the Golgi apparatus can be enhanced by the use of nitrocellulose as an embedding medium. Fixation of 1.5 mm thick pieces of fresh tissue for 8 hr in: glycine, 1.7 gm; 15% formalin, 100 ml; HNO₃, conc., 0.5 ml, at pH 2.6 followed by rinsing in water, 4 hr in 1.5% AgNO₃, another rinse, and 2 hr reduction in 1.5% hydroquinone in 15% formalin. This staining procedure yields consistently good results for rat, rabbit, and human tissues. Low-viscosity nitrocellulose embedding is done by infiltrating at 56 C in 7% nitrocellulose for 0.5 hr, 15% for 4 hr, and 27% for 1 hr. The nitrocellulose is hardened 2 hr in chloroform, after which, sections as thin as 5 μ can be cut on a sliding microtome. Gold toning and counterstaining can be done with the tissue affixed to the slide. The Golgi apparatus is stained dark brown to black, and there is better preservation of cellular detail than in tissues processed in paraffin.     

Characterization of N-linked oligosaccharides by affinoblotting with concanavalin A-peroxidase and treatment of the blots with glycosidases

Glycoproteins which bind concanavalin A (Con A) can be located on nitrocellulose sheets after electrophoretic transfer from slab gels, by sequential incubation of the sheets with Con A and peroxidase, and visualization of the peroxidase by an insoluble reaction product. We refer to this method as affinoblotting. Differential elution of Con A from the blots by washing the sheets with different concentrations of alpha-methylglycosides is used to demonstrate the affinity of Con A for the oligosaccharide side chains, and to differentiate between proteins with weak and those with high affinity for Con A. Concanavalin A has a high affinity for the four plant glycoproteins (phaseolin, phytohemagglutinin, jackbean alpha-mannosidase, and the glycosylated precursor of Con A) studied here. Incubation of the blots with alpha-mannosidase and endoglycosidase H (endo H) is used to demonstrate that the oligosaccharide chains can be degraded by glycosidases while the proteins are immobilized on the nitrocellulose. With this approach we show here that the four plant glycoproteins used as models in this study interact with Con A through high-mannose oligosaccharide side chains sensitive to alpha-mannosidase and endo H degradation.     

Use of nitrocellulose membranes as a scaffold in cell culture

Nitrocellulose membranes, one of the most important and oldest cellulose derivatives, are commonly used for nucleic acid and protein detection in research and diagnostic applications. However, a limited number of studies have explored whether they can act as scaffolds for cell growth. In this study, we investigated this polymeric material for its ability to support the growth of human cells. Eight established cell lines were examined for adherence, growth, spread, and survival on nitrocellulose membranes by optical microscopy after hematoxylin and eosin and/or immunocytochemical staining and by scanning electron microscopy. Apoptosis and leakage of lactate dehydrogenase (LDH) were also assessed. All cells readily adhered to and spread on the surface of nitrocellulose membranes as well as coverslips, and the cells maintained the expression of digestive system-specific genes. No significant change was detected in apoptosis or leakage of LDH from cells grown on nitrocellulose membranes. These results suggested that nitrocellulose membranes have a suitable cytocompatibility towards human cells and that they might be used for tissue-engineering scaffolds. Moreover, we demonstrate an additional and underused property of nitrocellulose of specific relevance to microscopic imaging, as it can be rendered virtually transparent, thus the cells growing on such membranes can be observed directly under an optical microscope after staining.     

Cross-blot and cross-dot system: a high-performance system for the detection of antigen-antibody complexes on nitrocellulose

We present a reliable, simple, and quick system for screening antibody-antigen complexes on nitrocellulose. The apparatus necessary for this system is inexpensive and easy to use, and it can be adapted to blot or dot analysis without any modification. The number of antibody-antigen combinations that can be tested in one experiment ranges from 25 to 31 for blot analysis and from 345 to 600 for dot analysis. This system also offers numerous experimental advantages: it makes it possible to estimate with only one experiment the contribution of the different reaction stages to background noise and so allows unambiguous interpretation of the antibody-antigen reaction. Furthermore, this system can be used for any hybridization experiment on nitrocellulose.     

组织培养中畸形胚状体及超度含水态苗的研究

畸形胚状和超度含水态苗是组织培养中常见的2种形态、生理异常现象,由于其难以发育成苗或很难移栽成活而严重影响组织培养的成功率。概述了这2种畸形现象的的形态特征及防止方法等,并讨论了应进一步研究的问题。     

Anatomical Changes and Immunolocalization of Cellulase during Abscission as Observed on Nitrocellulose Tissue Prints

A fundamental event in abscission is the breakdown of cell wall material in a discrete zone of cells known as the separation layer. Three dimensional images produced by viewing tissue prints of abscission zones on nitrocellulose (NC) membranes with incident illumination showed changes in the tissue integrity taking place in the separation layer as the process of abscission proceeded. The cell softening which occurs due to the dissolution of the cell wall appeared in the tissue prints as a diffuse line at the anatomical transition between the pulvinus and petiole and was easily observed on NC tissue prints of either longitudinal or serial cross-sections through abscission zones. In bean leaf abscission the dissolution of cell walls has been correlated with the appearance of a form of cellulase with an isoelectric point of pH 9.5. Antibodies specific for this enzyme were used to study the localization of 9.5 cellulase in the distal abscission zone of Phaseolus vulgaris L., cv Red Kidney after tissue printing on NC. It was found that 9.5 cellulase was localized in the separation layer but also occurred in the vascular tissue of the adjacent pulvinus. No antibody binding was observed in nonabscising tissue or preimmune controls. These results confirm previous biochemical studies and demonstrate that immunostaining of nitrocellulose tissue prints is a fast and reliable method to localize proteins or enzymes in plant tissue.     

Blotting intact immunoglobulins and other high-molecular-weight proteins after composite agarose-polyacrylamide gel electrophoresis

A composite agarose-polyacrylamide gel containing urea and sodium dodecyl sulfate reliably resolved unreduced human immunoglobulins according to their molecular weight. Intact immunoglobulins and a number of other macromolecules were readily transferred to nitrocellulose paper by either capillary or electrophoretic blotting, although the latter technique was more effective. Conventional antigen probing as well as immobilized antibody studies can be performed on the nitrocellulose transfers.     

Biotinylated derivative of a human brain lectin: synthesis and use in affinoblotting for endogenous ligand studies

Coupling of biotin to an endogenous lectin yields a probe which can be used for selective nonradioactive detection of complementary endogenous ligands. To exemplify practical applications of this type of compounds, we have synthesized and characterized a biotinylated derivative of a beta-galactoside-specific human brain lectin. Proteins which bind this lectin can be located on nitrocellulose sheets after electrophoretic transfer from gradient polyacrylamide gels, by sequential incubation with biotinylated probes and streptavidin-peroxidase, with visualization by an insoluble reaction product (affinoblotting). Biotinylated galactoside-binding plant lectins were used in the same way to visualize human brain glycoproteins, and their binding specificity was compared with that of human brain lectin. The results obtained by means of these different probes showed the usefulness of the endogenous lectin derivative to actually identify its endogenous partners. Thus this approach may find extended applications in the study of biological activities of vertebrate lectins in homologous systems, i.e., with lectins and ligands coming from the same tissue origin.     

Reproductive barrier and genomic imprinting in the endosperm of flowering plants

In flowering plants, success or failure of seed development is determined by various genetic mechanisms. During sexual reproduction, double fertilization produces the embryo and endosperm, which both contain maternally and paternally derived genomes. In endosperm, a reproductive barrier is often observed in inter-specific crosses. Endosperm is a tissue that provides nourishment for the embryo within the seed, in a similar fashion to the placenta of mammals, and for the young seedling after germination. This review considers the relationship between the reproductive barrier in endosperm and genomic imprinting. Genomic imprinting is an epigenetic mechanism that results in mono-allelic gene expression that is parent-of-origin dependent. In Arabidopsis, recent studies of several imprinted gene loci have identified the epigenetic mechanisms that determine genomic imprinting. A crucial feature of genomic imprinting is that the maternally and paternally derived imprinted genes must carry some form of differential mark, usually DNA methylation and/or histone modification. Although the epigenetic marks should be complementary on maternally and paternally imprinted genes within a single species, it is possible that neither the patterns of epigenetic marks nor expression of imprinted genes are the same in different species. Moreover, in hybrid endosperm, the regulation of expression of imprinted genes can be affected by upstream regulatory mechanisms in the male and female gametophytes. Species-specific variations in epigenetic marks, the copy number of imprinted genes, and the epigenetic regulation of imprinted genes in hybrids might all play a role in the reproductive barriers observed in the endosperm of interspecific and interploidy crosses. These predicted molecular mechanisms might be related to earlier models such as the "endosperm balance number" (EBN) and "polar nuclei activation" (PNA) hypotheses.     

What Are Imprinted Genes Doing in the Brain?

As evidence for the existence of brain?expressed imprinted genes accumulates, we need to address exactly what they are doing in this tissue, especially in terms of organizational themes and the major challenges posed by reconciling imprinted gene action in brain with current evolutionary theories attempting to explain the origin and maintenance of genomic imprinting. We are at the beginning of this endeavor and much work remains to be done but already it is clear that imprinted genes have the potential to influence diverse behavioral processes via multiple brain mechanisms. There are also grounds to believe that imprinting may contribute to risk of mental and neurological disease. As well as being a source of basic information about imprinted genes in the brain (e.g., via the newly established website, www.bgg.cardiff.ac.uk/imprinted_tables/index.html), we have used this chapter to identify and focus on a number of key questions. How are brain?expressed imprinted genes organized at the molecular and cellular levels? To what extent does imprinted action depend on neurodevelopmental mechanisms? Do imprinted gene effects interact with other epigenetic influences, especially early on in life? Are imprinted effects on adult behaviors adaptive or just epiphenomena? If they are adaptive, what areas of brain function and behavior might be sensitive to imprinted effects? These are big questions and, as shall become apparent, we need much more data, arising from interactions between behavioral neuroscientists, molecular biologists and evolutionary theorists, if we are to begin to answer them.     

A novel approach for identifying candidate imprinted genes through sequence analysis of imprinted and control genes

Through the sequence analysis of 27 imprinted human genes and a set of 100 control genes we have developed a novel approach for identifying candidate imprinted genes based on the differences in sequence composition observed. The imprinted genes were found to be associated with significantly reduced numbers of short interspersed transposable element (SINE) Alus and mammalian-wide interspersed repeat (MIR) repeat elements, as previously reported. In addition, a significant association between imprinted genes and increased numbers of low-complexity repeats was also evident. Numbers of the Alu classes AluJ and AluS were found to be significantly depleted in some parts of the flanking regions of imprinted genes. A recent study has proposed that there is active selection against SINE elements in imprinted regions. Alternatively, there may be differences in the rates of insertion of Alu elements. Our study indicates that this difference extends both upstream and downstream of the coding region. This and other consistent differences between the sequence characteristics of imprinted and control genes has enabled us to develop discriminant analysis, which can be used to screen the genome for candidate imprinted genes. We have applied this function to a number of genes whose imprinting status is disputed or uncertain.     

Large-scale analysis of 73 329 physcomitrella plants transformed with different gene disruption libraries: production parameters and mutant phenotypes

Gene targeting in the moss Physcomitrella patens has created a new platform for plant functional genomics. We produced a mutant collection of 73 329 Physcomitrella plants and evaluated the phenotype of each transformant in comparison to wild type Physcomitrella. Production parameters and morphological changes in 16 categories, such as plant structure, colour, coverage with gametophores, cell shape, etc., were listed and all data were compiled in a database (mossDB). Our mutant collection consists of at least 1804 auxotrophic mutants which showed growth defects on minimal Knop medium but were rescued on supplemented medium. 8129 haploid and 11 068 polyploid transformants had morphological alterations. 9 % of the haploid transformants had deviations in the leaf shape, 7 % developed less gametophores or had a different leaf cell shape. Other morphological deviations in plant structure, colour, and uniformity of leaves on a moss colony were less frequently observed. Preculture conditions of the plant material and the cDNA library (representing genes from either protonema, gametophore or sporophyte tissue) used to transform Physcomitrella had an effect on the number of transformants per transformation. We found correlations between ploidy level and plant morphology and growth rate on Knop medium. In haploid transformants correlations between the percentage of plants with specific phenotypes and the cDNA library used for transformation were detected. The number of different cDNAs present during transformation had no effect on the number of transformants per transformation, but it had an effect on the overall percentage of plants with phenotypic deviations. We conclude that by linking incoming molecular, proteome, and metabolome data of the transformants in the future, the database mossDB will be a valuable biological resource for systems biology.     

Detection of the low density lipoprotein (LDL) receptor on nitrocellulose paper with colloidal gold-LDL conjugates

Gold-low density lipoprotein (LDL) conjugates were used to detect the LDL receptor on nitrocellulose paper. Solubilized rat liver membrane proteins were subjected to electrophoresis and electroblotted onto nitrocellulose paper. The receptor was then detected as a red band (within 10 min) by overlaying with the LDL conjugates. The coloration was prevented by unlabeled LDL, EDTA, and suramin but not by unlabeled HDL3. In the dot blot assay, detection with the colloidal gold-LDL conjugates was as sensitive as both the autoradiographic method with 125I-labeled LDL and the biotinylated LDL method; the estimated limit of detection by scanning densitometry was 1.6 femtomoles of receptor protein. When the coloration obtained with the colloidal gold-LDL conjugates was intensified by photochemical silver staining, down to 200 attomoles of the LDL receptor could be detected. In this assay, the EDTA-sensitive binding of colloidal gold-LDL to solubilized hepatic membrane proteins was 12 times higher for rats treated with 17 alpha-EE than for normal rats. The use of colloidal gold-LDL conjugates is therefore a very easy, safe, inexpensive, fast and sensitive method for the detection of the LDL receptor on nitrocellulose paper. Furthermore, with silver staining and scanning densitometry, the colloidal gold-LDL conjugates could be used in a dot blot assay to quantify tissue and cell LDL receptors down to attomolar levels.     

A simple immunological detection method for the direct screening of genes from clone banks

A simple immunoassay has been developed which can be used in the isolation of particular gene(s) from a clone bank of recombinant plasmids. A clone bank of the DNA is constructed with a plasmid vector and maintained in Escherichia coli. The recombinant clones were filtered onto a hydrophobic grid membrane and grown up into individual colonies, and a replica was made onto nitrocellulose paper. The bacterial cells were then lysed with chloroform and the proteins were immobilized onto the nitrocellulose paper. The nitrocellulose paper is then reacted with a rabbit antibody preparation made against the particular antigenic product to detect the recombinant clone which carries the corresponding gene. The bound antibodies can be detected easily by a colorimetric assay using goat anti-rabbit antibodies conjugated to horseradish peroxidase. Positively reacting clones can be recovered from the master hydrophobic grid membrane filter for further characterization. We proposed to call this method "colony ELISA blot" and described the isolation of the genes coding for the soluble antigens of Pasteurella haemolytica using this method.     

Composite matrix membranes as synthetic receptor systems. 2. Structural-morphological characteristics

Structural and morphological characteristics of composite imprinted membranes for selective recognizing of adenosine 3',5'-cyclic monophosphate (cAMP) were studied. Composite polyvinylidene fluoride microfiltration membranes (Millipore) covered with a thin imprinted polymer layer were prepared using photoinitiated copolymerization of dimethylaminoethylmetacrylate with trimethylolpropanethrimethacrylate in the presence of cAMP as template. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) were used to visualise surfaces and cross-sections of imprinted membranes and to determine their structural and morphological parameters such as pore size, thickness of selective imprinted layer, surface roughness as well as total surface contact area. The impact of structural characteristics on separation properties of the imprinted membranes was analyzed. It was found out that the thickness of the imprinted polymer layer with optimal recognizing properties is limited.     

Neoglycoenzymes: a versatile tool for lectin detection in solid-phase assays and glycohistochemistry

Carbodiimide-mediated coupling of p-aminophenyl glycosides to a naturally nonglycosylated enzyme yields a neoglycoenzyme. This compound combines inherent enzymatic activity with synthetically conferred ligand properties to lectins. Appropriate choice of the ligand allows custom-made synthesis to reliably detect various types of lectins. To exemplify practical applications of this class of compounds, glycosylated bacterial beta-galactosidase has been employed to quantitate plant lectins, immobilized on plastic surfaces as well as on nitrocellulose. Competitive inhibition by specific sugar ascertained the dependence of binding on protein--carbohydrate interactions. In view of lectins as tools, a sandwich lectin-binding assay for high mannose-type glycoprotein detection has been modified to principally facilitate wide application to other lectin-reactive sugar chains by introducing the neoglycoenzyme. In addition to lectin determination in solid-phase assays, neoglycoenzymes allow one to glycohistochemically localize endogenous lectins in tissue prints and tissue sections with a minimum number of steps. This nonradioactive, rapid, sensitive, and convenient assay concept, based on conjugation of a ligand to an enzyme with maintenance of its receptor-binding activity, may find extended application beyond lectinology in receptor analysis.