Multivalent Formation Research Articles

Photoclickable Dendritic Molecular Glue: Noncovalent-to-Covalent Photochemical Transformation of Protein Hybrids

A water-soluble dendron with a fluorescein isothiocyanate (FITC) fluorescent label and bearing nine pendant guanidinium ion (Gu(+))/benzophenone (BP) pairs at its periphery (Glue(BP)-FITC) serves as a "photoclickable molecular glue". By multivalent salt-bridge formation between Gu(+) ions and oxyanions, Glue(BP)-FITC temporarily adheres to a kinesin/microtubule hybrid. Upon subsequent exposure to UV light, this noncovalent binding is made permanent via a cross-linking reaction mediated by carbon radicals derived from the photoexcited BP units. This temporal-to-permanent transformation by light occurs quickly and efficiently in this preorganized state, allowing the movements of microtubules on a kinesin-coated glass plate to be photochemically controlled. A fundamental difference between such temporal and permanent bindings was visualized by the use of "optical tweezers".

Friction-Mediated Dynamic Disordering of Phospholipid Membrane by Mechanical Motions of Photoresponsive Molecular Glue: Activation of Ion Permeation

A water-soluble photoresponsive molecular glue, Azo-(18)Glue, consisting of a photochromic azobenzene core and two adhesive dendritic wedges with a total of 18 peripheral guanidinium ion (Gu(+)) pendants tightly adheres to the surface of a phospholipid membrane, even in buffer, via a multivalent salt-bridge formation with phosphate anions. A photomechanical motion of adhering Azo-(18)Glue possibly gives rise to dynamic structural disordering of the phospholipid membrane and activates transmembrane ion permeation. In sharp contrast, no activation of ion permeation results when poorly adhesive Azo-(6)Glue carrying only six Gu(+) pendants is used in place of Azo-(18)Glue.

Chromosome number, meiotic behavior and pollen fertility of six species ofChrysolaena(Vernonieae, Asteraceae)

Chromosome number, meiotic behavior and pollen fertility were determined for six species of the genus Chrysolaena H.Rob. (Vernonieae, Asteraceae). All the species had a base chromosome number of x = 10, with somatic numbers that ranged between 2n = 20 and 2n = 60. The chromosome number of Chrysolaena obovata (Less.) Dematt. (2n = 4x = 40) and a new cytotype for C. lithospermifolia (Hieron.) H.Rob. (2n = 4x = 40) are reported for the first time. Normal meiotic behavior was observed for the diploid species C. platensis (Spreng.) H. Rob., C. propinqua (Hieron.) H. Rob. and C. flexuosa (Sims) H. Rob. However, the polyploid taxa C. obovata, C. lithospermifolia (tetraploid) and C. cognata (Less.) Dematt. (hexaploid) showed many meiotic irregularities such as multivalent formation, chromosomes outside plate, early segregation of chromosomes, laggards and bridges in anaphase and telophase. Pollen fertility was high in all species, however, including in the polyploids. Polyploidy is more common in Chrysolaena than...

Chiral structure of F-actin bundle formed by multivalent counterions

The mechanism of multivalent counterion-induced bundle formation by filamentous actin (F-actin) is studied using a coarse-grained model and molecular dynamics simulation. Real diameter size, helically ordered charge distribution and twist rigidity of F-actin are taken into account in our model. The attraction between parallel F-actins induced by multivalent counterions is studied in detail and it is found that the maximum attraction occurs between their closest charged domains. The model F-actins aggregate due to the like-charge attraction and form closely packed bundles. Counterions are mostly distributed in the narrowest gaps between neighboring F-actins inside the bundles and the channels between three adjacent F-actins correspond to low density of the counterions. Density of the counterions varies periodically with a wave length comparable to the separation between consecutive G-actin monomers along the actin polymers. Long-lived defects in the hexagonal order of F-actins in the bundles are observed that their number increases with increasing the bundles size. Combination of electrostatic interactions and twist rigidity has been found not to change the symmetry of F-actin helical conformation from the native 13/6 symmetry. Calculation of zero-temperature energy of hexagonally ordered model F-actins with the charge of the counterions distributed as columns of charge domains representing counterion charge density waves has shown that helical symmetries commensurate with the hexagonal lattice correspond to local minima of the energy of the system. The global minimum of energy corresponds to 24/11 symmetry with the columns of charge domains arranged in the narrowest gaps between the neighboring F-actins.

Impact of Reciprocal Translocations and Non-Synchronous Disjunction of Chromosomes on Pollen Fertility in <i>Astragalus chlorostachys</i> from the Northwest Himalayas (India)

Summary Cytological investigations carried out on a population basis in Astragalus chlorostachys Lindl. which exists at diploid level (n=8) revealed the presence of quadrivalents, the late disjunction of 1–4 bivalents and some pollen sterility (34%) in 1 accession collected from Koksar, at 3,140 m in the Lahaul Valley (Lahaul-Spiti, Himachal Pradesh). The presence of quadrivalents in this diploid species seems to be a consequence of reciprocal translocations. The occurrence of structural heterozygosity in the species has been reported here for the first time. All of the other populations showed normal bivalent formation and regular sporad generation and 100% pollen fertility. The paper here discusses in detail the course of meiosis and the effect of multivalent formation and non-synchronous disjunction of bivalents on meiotic course and pollen fertility. Astragalus chlorostachys Lindl., known variously as Astragalus altissimus Rech. f., Astragalus nuristanicus Kitam. and Hedysarum strobiliferum Baker, belongs to the tribe Galegeae of the family Fabaceae. It is a tall woody herb with sparsely pubescent stems. Leaves are imparipinnately compound and with leaflets oblong, obtuse and glabrescent. Yellowish-white flowers are produced in axillary, peduncled racemes. The species is native to Afghanistan, Bhutan, India, Nepal and Pakistan. In India it grows in moist shady places between altitudes of 2,000–3,600 m in the states of Assam, Himachal Pradesh, Jammu-Kashmir, Sikkim and Uttarakhand (Chowdhery and Wadhwa 1984, Naithani 1984, Sharma and Jamwal 1988, Wenninger 1991, Sanjappa 1992). Local people in the Kumaon Himalayas use a whole plant infusion as a tonic and febrifuge; decoction of the root is given in tuberculosis (Trivedi 2006). During cytomorphological surveys of the species in various cold regions of the Northwest Himalayas, the authors have detected 1 population which displayed the presence of multivalents, late disjunction of some bivalents and some pollen sterility. All of the other populations showed normal bivalent formation and regular sporad generation and 100% pollen fertility. In this paper, the aim was to study in detail the male meiotic course and to investigate the effect of multivalent formation and non-synchronous disjunction of bivalents on meiotic course and pollen fertility. Materials and methods Materials for male meiotic studies were collected from the different localities falling in the

Synthesis and cytological analyses of hybrids between hexaploid wheat, with and without Ph1, and diploid wheatgrass

The usefulness of wide hybridization in genetic enhancement of crop plants like wheat, Triticum aestivum L. (2n = 6x = 42; AABBDD genomes), is well documented. Diploid wheatgrass, Lophopyrum elongatum (Host) A. Love (2n = 2x = 14; EE genome), is a source of several desirable traits including Fusarium head blight resistance. The objective of this study was to report on the synthesis and cytological analyses of wheat × wheatgrass hybrids and highlight homoeologous pairing involving the wheat and wheatgrass chromosomes. We hybridized wheatgrass with two important, crossable cultivars, ‘Chinese Spring,’ and ‘Fukohokomugi,’ of bread wheat and produced 11 tetraploid (2n = 4x = 28; ABDE) and two pentaploid (2n = 5x = 28; ABDEE) hybrids. To help promote homoeologous pairing and hence genetic recombination between the wheat and grass chromosomes, we also used the Chinese Spring mutant ph1bph1b. Using molecular markers, we readily detected intergeneric hybrids without Ph1. The hybrids without Ph1 showed multivalent formation involving wheat and grass chromosomes. However, the lack of Ph1 produced a much higher homoeologous pairing in the haploid chromosome complement in the CS ph1bph1b × wheatgrass hybrids than in the disomic complement of CS ph1bph1b. The pentaploid hybrids resulted from the fusion of a normal 21-chromsome gamete of the wheat parent with an unreduced, 14-chromsome gamete of diploid wheatgrass. These hybrids showed a remarkable degree of Ph1-enforced preferential pairing among the homologous wheatgrass chromosomes. Varying degrees of homoeologous pairing in various hybrids were detected that could facilitate alien gene transfer into wheat.

SNAP Dendrimers: Multivalent Protein Display on Dendrimer‐Like DNA for Directed Evolution

Display systems connect a protein with the DNA encoding it. Such systems (e.g., phage or ribosome display) have found widespread application in the directed evolution of protein binders and constitute a key element of the biotechnological toolkit. In this proof-of-concept study we describe the construction of a system that allows the display of multiple copies of a protein of interest in order to take advantage of avidity effects during affinity panning. To this end, dendrimer-like DNA is used as a scaffold with docking points that can join the coding DNA with multiple protein copies. Each DNA construct is compartmentalised in water-in-oil emulsion droplets. The corresponding protein is expressed, in vitro, inside the droplets as a SNAP-tag fusion. The covalent bond between DNA and the SNAP-tag is created by reaction with dendrimer-bound benzylguanine (BG). The ability to form dendrimer-like DNA straightforwardly from oligonucleotides bearing BG allowed the comparison of a series of templates differing in size, valency and position of BG. In model selections the most efficient constructs show recoveries of up to 0.86 % and up to 400-fold enrichments. The comparison of mono- and multivalent constructs suggests that the avidity effect enhances enrichment by up to fivefold and recovery by up to 25-fold. Our data establish a multivalent format for SNAP-display based on dendrimer-like DNA as the first in vitro display system with defined tailor-made valencies and explore a new application for DNA nanostructures. These data suggest that multivalent SNAP dendrimers have the potential to facilitate the selection of protein binders especially during early rounds of directed evolution, allowing a larger diversity of candidate binders to be recovered.

Polyploidization increases meiotic recombination frequency in Arabidopsis

BackgroundPolyploidization is the multiplication of the whole chromosome complement and has occurred frequently in vascular plants. Maintenance of stable polyploid state over generations requires special mechanisms to control pairing and distribution of more than two homologous chromosomes during meiosis. Since a minimal number of crossover events is essential for correct chromosome segregation, we investigated whether polyploidy has an influence on the frequency of meiotic recombination.ResultsUsing two genetically linked transgenes providing seed-specific fluorescence, we compared a high number of progeny from diploid and tetraploid Arabidopsis plants. We show that rates of meiotic recombination in reciprocal crosses of genetically identical diploid and autotetraploid Arabidopsis plants were significantly higher in tetraploids compared to diploids. Although male and female gametogenesis differ substantially in meiotic recombination frequency, both rates were equally increased in tetraploids. To investigate whether multivalent formation in autotetraploids was responsible for the increased recombination rates, we also performed corresponding experiments with allotetraploid plants showing strict bivalent pairing. We found similarly increased rates in auto- and allotetraploids, suggesting that the ploidy effect is independent of chromosome pairing configurations.ConclusionsThe evolutionary success of polyploid plants in nature and under domestication has been attributed to buffering of mutations and sub- and neo-functionalization of duplicated genes. Should the data described here be representative for polyploid plants, enhanced meiotic recombination, and the resulting rapid creation of genetic diversity, could have also contributed to their prevalence.

Occurrence of differential meiotic associations and additional chromosomes in the embryo-sac mother cells of Allium roylei Stearn

A small population of complex translocation heterozygote plants of Allium roylei from the Bani region of Jammu Province was studied for meiosis in the female track. This study resulted in identification of two variants, having embryo-sac mother cells (EMCs) with more than 16 chromosomes. EMCs of the remaining plants invariably had diploid (2n = 16) chromosome complement. Female meiosis, in general, was found to be abnormal, with nearly 23% and 11% chromosomes associating as quadrivalents or trivalents at prophase I and at metaphase I, respectively. This was followed by irregular segregation of chromosomes at anaphase I. Amongst the variants; one had 38% EMCs with eight bivalents plus two small sized chromosomes. Their small size, dispensable nature and tendency to affect the pairing behaviour of normal complement are some of the features that latter chromosomes share with the B chromosomes. Seventeen to nineteen chromosomes were observed in 35% EMCs of other variant; the remaining cells had 16 chromosomes. Chromosomal behaviour in both kind of cells (euploid and aneuploid) was more or less similar. Unlike female meiocytes, male meiocytes analysed earlier of this strain always had 16 chromosomes which paired to form extremely complex associations involving 3-16 chromosomes. The most likely cause of this asynchrony with regards to number of chromosomes involved in multivalent formation seems to be interaction of genes controlling chiasma formation with the different physiological conditions of male and female meiocytes.

Development of an Interspecific Hybrid (Triploid) by Crossing Gossypium hirsutum and G. arboreum

The 2 species G. hirsutum and G. arboreum were crossed and triploid hybrid (AAD) was developed as a first step of crossing. The triploid is sterile and its hybridity was confirmed with morphological traits and DNA based SSR markers. The chromosome analysis at metaphase-I exhibited 13AA bivalents and 13D univalents at most of the PMC's studied which also confirmed its triploid nature. The mean chromosome pairing association was recorded as 12.90% (univalent), 10.45% (bivalent), 0.45% (trivalent), and 0.48% (tetravalent). The formation of multivalents depicted genome association between 2 species. The further utility of synthesized triploid with respect to the development of CLCuV resistant introgression genotypes is discussed

Meiotic behavior of pollen mother cells in relation to ploidy level of somatic hybrids between Solanum tuberosum and S. chacoense

Potato somatic hybrids obtained by protoplast fusion between Solanum tuberosum (4x) and Solanum chacoense (2x) were investigated for genome stability and meiotic behavior associated with the pollen viability in order to elucidate the mechanism influencing the fertility of the somatic hybrids. The ploidy level detections conducted in 2004 and 2007 demonstrated that 68 out of 108 somatic hybrids had their ploidy level changed to be uniform and euploidy after successive in vitro subcultures, which mainly occurred in octaploids, aneuploids, and mixoploids, while 74% hexaploids were still stable in their genome dosage in 2007. Different types of abnormal meiotic behavior were observed during the development of pollen mother cells (PMCs) including the formation of univalents, multivalents, laggard chromosomes, and chromosomal bridges, as well as triads and polyads. A higher proportion of abnormal meiosis seemed to be accompanied with a genome dosage higher than the hexaploids expected in this study. A significant positive correlation between defective PMCs and the number of small pollen grains and negative correlation between number of small pollen grains and pollen viability strongly suggested that abnormal meiosis could be a causal factor influencing the fertility of the somatic hybrids. The hexaploids with stable genome dosage and a certain level of fertility will have great potential in a potato breeding program.

Isolation of a Novel Nanobody Against HER-2/neuUsing Phage Displays Technology

In 1993, Hamers-Casterman and colleagues discovered a novel type of IgG antibodies.1 Camelid serum contains functional heavy-chain homodimeric antibodies without light chains. The variable domain of these single chain antibodies are named as VHHs (nanobodies). The binding ability of VHH to antigens was found to be similar or even better than classical IgG.2 Single-domain antibody was also identified in special cartilaginous fish by Greenberg and colleagues.3 It was also found that they are suitable for expression as multivalent formats like bispecific, enzyme, or toxin-VHH fusions to increase avidity without using current linkers like those that existed between VL and VH domains, which often result in aggregation and decrease affinity because of mispairing of VL and VH domains.4 Lacking of the mispairing problems allows designing more flexible linkers and successfully achieving functional trivalent-bispecific VHHs.5,6 Sequencing and crystallographic analysis of camel VHH has showed homology to the human VHH. The above facts prepare some advantages for biotechnological applications of VHH, such as facile genetic manipulation, increased functional size of libraries, high physicochemical stability, ease of production of multivalent forms, high stability, recognition of hidden antigenic sites, well expression, rapid tissue penetration, and fast blood clearance.7 Soluble VHHs have been produced in Escherichia coli (E. coli), filamentous fungi, Saccharomyces cerevisiae, and Pichia pastoris. 8–11 VHH single-domains can be used whenever high stability is required, such as use in shampoo for preventing dandruff.12 They were used in immunoaffinity purification as capturing reagents and in biosensor technology.13,14 VHH is also reported to be a good candidate for oral immunotherapy because of its resistance against high pH, and its ability to bind to the target in the presence of high concentrations of many other agents.15 This type of functional VHH antibody …

Genetic, embryonic and anatomical study of an interspecific cassava hybrid

A molecular, anatomical and cytogenetic study of an interspecific hybrid between Manihot esculenta (cassava) and the wild species M. oligantha was carried out. Cytogenetics revealed relatively complete chromosome pairing and high viability of the pollen grains. Ovule structure examined by the clearing method showed polyembryony in 2.7% of the ovules. Doubling of the chromosome number resulted in an increase in polyembryony of up to 18% and a reduction in pollen viability. Multivalent formation was also observed. An anatomical study of stems of diploid and tetraploid hybrids showed a larger number of vascular bundles in the tetraploid type.

Adapter-Directed Display: A Modular Design for Shuttling Display on Phage Surfaces

A novel adapter-directed phage display system was developed with modular features. In this system, the target protein is expressed as a fusion protein consisting of adapter GR1 from the phagemid vector, while the recombinant phage coat protein is expressed as a fusion protein consisting of adapter GR2 in the helper phage vector. Surface display of the target protein is accomplished through specific heterodimerization of GR1 and GR2 adapters, followed by incorporation of the heterodimers into phage particles. A series of engineered helper phages were constructed to facilitate both display valency and formats, based on various phage coat proteins. As the target protein is independent of a specific phage coat protein, this modular system allows the target protein to be displayed on any given phage coat protein and allows various display formats from the same vector without the need for reengineering. Here, we demonstrate the shuttling display of a single-chain Fv antibody on phage surfaces between multivalent and monovalent formats, as well as the shuttling display of an antigen-binding fragment molecule on phage coat proteins pIII, pVII, and pVIII using the same phagemid vectors combined with different helper phage vectors. This adapter-directed display concept has been applied to eukaryotic yeast surface display and to a novel cross-species display that can shuttle between prokaryotic phage and eukaryotic yeast systems.

Carbohydrate arrays: recent developments in fabrication and detection methods with applications

Carbohydrate arrays can provide facile analyses of carbohydrate-binding proteins, antibodies in serum, and enzyme activities with minimal quantities of sugar samples. Recent developments in detection methods for carbohydrate arrays, especially surface plasmon resonance (SPR) and mass spectrometry, have enabled not only qualitative but in some cases quantitative data analysis in a variety of multivalent display formats. These advancements make diagnostic tools based on carbohydrate arrays more promising for infectious disease detection, cancer monitoring, and vaccine development.

Fluorous-Based Small-Molecule Microarrays for Protein, Antibody and Enzyme Screening

Microarray techniques based on covalent and noncovalent compound immobilization have been developed for screening proteins, antibodies and enzymes to probe the possible biological roles of these interactions as well as their therapeutic and diagnostic potential. Small-molecule microarrays are particularly valuable for creating and probing multivalent displays of molecules, such as saccharides that mimic the multivalent displays of cell surface-bound compounds. After development of the method for the screening of carbohydrates in a multivalent display format, microarrays based on noncovalent fluorous interactions have seen use in probing protein-binding partners with a range of arrayed compounds. In this article, existing strategies and future perspectives for fluorous-based small-molecule microarrays for protein, antibody and enzyme screening will be presented. To date, qualitative and quantitative fluorous-based microarrays have offered important information regarding biomolecular interactions. Larger compound arrays created with automated fluorous-based synthesis and diagnostic tools based on fluorous-based microarrays are likely ahead.

Glycan arrays: recent advances and future challenges.

Carbohydrate arrays, also referred to as glycan arrays, are composed of various oligosaccharides and/or polysaccharides immobilized on a solid support in a spatially defined arrangement. This technology provides a powerful, high-throughput approach to examining carbohydrate-macromolecule interactions, and glycan arrays have had a significant impact on the field of glycobiology. This review focuses on recent advances in glycan array technology, limitations, and opportunities for improvement. In particular, new methods for the production of natural glycan arrays and chemoenzymatic approaches are greatly expanding the diversity of structures on arrays. Since multivalent complex formation is generally required to achieve tight binding, methods to evaluate and modulate presentation are vital for enhancing the capabilities of this technology.

CYTOLOGICAL PECULIARITIES IN TOMATO (SOLANUM LYCOPERSICUM) NUCLEAR MALE STERILITY

A cytological investigation for identification of the meiotic stages and mechanism of male sterility expression in nuclear male sterile tomato line No. 6944aa (msms1035) was performed in “Maritsa” Vegetable Crops Research Institute, Plovdiv, Bulgaria. The male sterile genotypes from second backcross progenies (BC1P2 and BC3P2) of the hybrid between line No. 6944aa and line No. 1 were cytologically characterized. The purpose was – to study the heritage and stability of the male sterility in creation of new sterile tomato line. The meiotic analysis in the pollen mother cells of sterile genotypes from line No. 6944aa revealed, that 12 bivalents were formed during the diakinesis and metaphase one in 74.1% of the cells. In parallel with bivalents 1 to 2 quadrivalents were observed in 15.8% of PMC and in case of desynapsis and early bivalent and quadrivalent separation 2 to 22 univalents in 10.1% of the cells were established. The normal pairing of homologous chromosomes of sterile msms 1035 plants from the different backcross progenies was described in 73.6 and 75.9% of PMC. The similar formation of multivalents and univalets, as in the mother sterile plants, was observed in backcross msms 1035 plants. The chromosomes have been distributed visually regular toward the spindle poles during the anaphase one both in the mother and in the backcross sterile genotypes in about 91–92% of the meiocytes. The cells lethality took place after second telophase, as the expression of the gene responsible for cytokinesis was blocked and we did not observe tetrad stage and microspore formation in msms 1035 plants. The cytological mechanism of nuclear male sterility in tomato, determined by gene ms 1035 probably is the presence of reciprocal, symmetric heterozygous translocations, as the disruptions of chromosomes destroyed expression of the genes which defined normal initiation, running and termination of the some meiotic stages. As a result of meiotic abnormalities chromosomes and gene unbalanced nuclei were formed, which reflected in pollen mother cell lethality.

Cytological studies of the genus Rosa with special reference to the section Caninae.

The Caninae type meiosis in the genus Rosa is demonstrated in a hexaploid, Rosa jundzillii Bess. (2n = 42). A probable hybrid plant which is taxonomically identical with the jundzillii hexaploid was found to be heptaploid (2n = 49) with irregular contraction of chromosomes in the late prophase, prometaphase and metaphase. Multivalent-like configurations were also observed. This is a common feature in other Rosa hybrids, but expressed in different degrees. Of particular interest in the meiosis of Rosa are (1) the diffuse stage, (2) the irregularities in the condensation of chromosomes and the formation of multivalents and (3) the appearance and disappearance of nucleoli and nucleolus-like bodies.

Chromosomal polymorphism in isolated populations of Elymus (Agropyron) in the Aegean:

Elymus rechingeri is a tetraploid (2n = 28) cross-fertilizer which grows in small isolated populations mainly on the Aegean islands. Chromosome morphology was studied in plants raised from seeds collected in nature and in plants resulting from inter- and intrapopulation crosses as well as selfing. Chromosomal polymorphism predominated in the material originating from nature and was observed both between and within populations. This polymorphism was expressed in the variable appearance of the three pairs of satellite chromosomes. The most common constitution of satellite chromosomes was found in Aegean and Egyptian materials and was considered to be the constitution characteristic of the species. Expected combinations of different types of satellite chromosomes were found in the offspring after inter- and intrapopulation crosses and after selfing. Some offspring plants also showed structural and numerical chromosome deviations. Fertility was lower in crosses between than within populations indicating genetical differences between populations. The karyotype of E. rechingeri shows great similarities to the karyotypes of E. furctus ssp. boreo-atlanticus (2n = 28) and E. striatulus (2n = 14). The genome formula J1J1J3J3 was suggested for E. rechingeri. Meiosis was generally normal but asynapsis and multivalent formation at metaphase I and separation difficulties at anaphase I were also encountered.