Multivalent Association Research Articles

ETO2 Recruitment of NuRD to the γ-Globin Locus Involves Multivalent Interaction with GATAD2A

Increasing the expression of fetal hemoglobin ameliorates the symptoms of sickle cell disease and β-thalassemia. Therefore, blocking fetal hemoglobin silencing in adult erythrocytes represents a potent strategy for treating these diseases. Extensive research over the past five decades have uncovered critical transcription factors involved in silencing fetal hemoglobin, including Bcl11A and Zbtb7A (LRF), among others. The Nucleosome Remodeling and Deacetylase (NuRD) complex represents a central co-regulatory factor recruited by these proteins and DNA methylation to silence gene expression. Hence, NuRD represents a potential target for blocking silencing by multiple factors simultaneously, thereby leading to robust induction of fetal hemoglobin. We previously have shown that disrupting the MBD2-NuRD complex induces high levels of fetal hemoglobin in both the HUDEP-2 tissue culture cell line and primary patient-derived bone marrow stem cells [Yu et al. (2019) Haematologica, 104(12), 2361-2371]. Recent work by the Dean lab [Guo et al. (2020). Nucleic Acids Research, 48(18), 10226-10240] added to the list of factors that bind and recruit NuRD to the globin locus. They showed that ETO2, a well-established factor in erythroid differentiation, binds directly to NuRD, which required the tetramerization domain of ETO2. However, the specific contacts driving this interaction remain unknown. Based on previous work by the Bushweller [Liu et al. (2007) Cancer Cell, 11(6), 483-497] and Vermeulen [Spruijt et al. (2016) Cell Reports, 17(3), 783-798] groups, we hypothesized that ETO2 binds directly to the GATAD2A component of NuRD through the Nervy Homology Region-4 (NHR4) domain of ETO2 and multiple polyproline-leucine (PPPL) motifs in GATAD2A. Here we use NMR analyses to show that the ETO2-NHR4 domain binds each of four polyproline-leucine motifs in GATAD2A. We measure binding by chemical shift analysis which reveals a weak affinity (KD ~200-300 μM) for the individual peptides. We then show that the ETO2-NHR3 domain contributes to weak homo-oligomerization (dimer-tetramer formation) by gel filtration analysis. Importantly, multivalent interaction between the PPPLx4 region of GATAD2A and a construct including both the NHR3 and NHR4 domains of ETO2 leads to a dramatic increase in binding affinity (KD ~ 4 μM) as measured by isothermal titration calorimetry. Furthermore, using an in-cell bioluminescent resonance energy transfer assay (NanoBRET), we show that including the NHR2 tetramerization domain augments association between these proteins. Mutating key residues in either GATAD2A-PPPLx4 or ETO2-NHR4 domains disrupts this association in cells. Finally, we show that enforced expression of a peptide from GATAD2A containing the four polyproline-leucine motifs consistently induces a low-level of fetal hemoglobin mRNA expression in K-562 cells. Together, these findings establish that the ETO2-NHR4 domain plays a central role in recruitment of NuRD for gene silencing. Tetramerization of the NHR2 and NHR3 domains leads to multivalent association with GATAD2A, which dramatically increases binding affinity. This result has mechanistic implications for fetal hemoglobin silencing and erythroid differentiation, potentially leading to a novel therapeutic target for treating β-hemoglobinopathies. Furthermore, gene rearrangements involving the ETO family of proteins drive the development of acute myeloid leukemia. Our studies provide an explanation for how the stoichiometry of the fusion proteins may dictate which co-regulatory complexes are brought to a specific site and suggest a strategy for selectively targeting these complexes.

Covalent and Oriented Immobilization of Antibodies through Systematic Modification of Photoactivatable RNA Hybrid Aptamers

We report a covalent and well-oriented strategy of immobilization of antibodies using photoactivatable Fc-binding RNA aptamers (FcBAs). We prepared several types of FcBAs that were further modified with photoaffinity probes (i.e., benzophenone or diazirine), and evaluated the binding capabilities and the photo-crosslinking efficiency of them via pull-down assays and fluorescence analyses, respectively. Initial moderate photo-crosslinking efficiency (Kd ∼ 110 nM) was substantially improved by multivalent association of FcBAs and structural modification of FcBAs. For a conceptual proof, covalent crosslinking of human IgG on FcBA-tethered solid chips was fabricated and investigated by scanometry, which eventually proved real applicability of the present scheme toward immunoassays.

Systematic simulation of the interactions of pleckstrin homology domains with membranes.

Pleckstrin homology (PH) domains can recruit proteins to membranes by recognition of phosphatidylinositol phosphate (PIP) lipids. Several family members are linked to diseases including cancer. We report the systematic simulation of the interactions of 100 mammalian PH domains with PIP-containing membranes. The observed PIP interaction hotspots recapitulate crystallographic binding sites and reveal a number of insights: (i) The β1 and β2 strands and their connecting loop constitute the primary PIP interaction site but are typically supplemented by interactions at the β3-β4 and β5-β6 loops; (ii) we reveal exceptional cases such as the Exoc8 PH domain; (iii) PH domains adopt different membrane-bound orientations and induce clustering of anionic lipids; and (iv) beyond family-level insights, our dataset sheds new light on individual PH domains, e.g., by providing molecular detail of secondary PIP binding sites. This work provides a global view of PH domain/membrane association involving multivalent association with anionic lipids.

Yorkie-Warts Complexes are an Ensemble of Interconverting Conformers Formed by Multivalent Interactions

WW domain and PPXY motif sequences are often presented in multiple copies in several proteins at the critical homeostasis juncture of cell death versus cell replication. While biophysical studies of single WW domain-single PPXY motif complexes abound in the literature, the molecular mechanisms of multivalent WW domain-PPXY assemblies are still poorly understood. by way of investigating such assemblies, we have characterized the multivalent association of the entire cognate binding domains, two WW domains and five PPXY motifs respectively, of the Yorkie transcription coactivator and the Warts tumor suppressor. Isothermal titration calorimetry and native-state mass spectrometry of Yorkie interactions with the full-length Warts PPXY domain, and numerous PPXY motif variants of Warts show that the two proteins assemble via binding of the tandem WW domains to adjacent PPXY pairs to produce an ensemble of interconverting complexes of variable stoichiometries, binding energetics and WW domain occupancy. Apparently, the Yorkie tandem WW domains first target the two adjacent PPXY motifs at the C- terminus of the Warts polypeptide and additional WW domains bind unoccupied motifs. The ensemble of interconverting conformers may be common in multivalent WW domain-PPXYinteractions, and may promote the adaptability and versatility of WW domain-PPXY mediated cellular processes.

Yorkie-Warts Complexes are an Ensemble of Interconverting Conformers Formed by Multivalent Interactions

Multiple copies of WW domains and PPXY motif sequences are often reciprocally presented by regulatory proteins that interact at crucial regulatory steps in the cell life cycle. While biophysical studies of single WW domain-single PPXY motif complexes abound in the literature, the molecular mechanisms of multivalent WW domain-PPXY assemblies are still poorly understood. By way of investigating such assemblies, we characterized the multivalent association of the entire cognate binding domains, two WW sequences and five PPXY motifs respectively, of the Yorkie transcription coactivator and the Warts tumor suppressor. Isothermal titration calorimetry, sedimentation velocity, size-exclusion chromatography coupled to multi-angle light scattering and native-state mass spectrometry of Yorkie WW domains interactions with the full-length Warts PPXY domain, and numerous PPXY motif variants of Warts show that the two proteins assemble via binding of tandem WW domains to adjacent PPXY pairs to produce an ensemble of interconverting complexes of variable stoichiometries, binding energetics and WW domain occupancy. Apparently, the Yorkie tandem WW domains first target the two adjacent PPXY motifs at the C-terminus of the Warts polypeptide and additional WW domains bind unoccupied motifs. Similar ensembles of interconverting conformers may be common in multivalent WW domain-PPXY interactions to promote the adaptability and versatility of WW domain-PPXY mediated cellular processes.

Lipid Bilayer-like Mixed Self-Assembled Monolayers with Strong Mobility and Clustering-Dependent Lectin Affinity.

Glycans at the surface of cellular membranes modulate biological activity via multivalent association with extracellular messengers. The lack of tuneable simplified models mimicking this dynamic environment complicates basic studies of these phenomena. We here present a series of mixed reversible self-assembled monolayers (rSAMs) that addresses this deficiency. Mixed rSAMs were prepared in water by simple immersion of a negatively charged surface in a mixture of sialic acid- and hydroxy-terminated benzamidine amphiphiles. Surface compositions derived from infrared reflection-absorption spectroscopy (IRAS) and film thickness information (atomic force microscopy, ellipsometry) suggest the latter to be statistically incorporated in the monolayer. These surfaces' affinity for the lectin hemagglutinin revealed a strong dependence of the affinity on the presentation, density, and mobility of the sialic acid ligands. Hence, a spacer length of 4 ethylene glycol and a surface density of 15% resulted in a dissociation constant Kd,multi of 1.3 × 10-13 M, on par with the best di- or tri-saccharide-based binders reported to date, whereas a density of 20% demonstrated complete resistance to hemagglutinin binding. These results correlated with ligand mobility measured by fluorescence recovery after photobleaching which showed a dramatic drop in the same interval. The results have a direct bearing on biological cell surface multivalent recognition involving lipid bilayers and may guide the design of model surfaces and sensors for both fundamental and applied studies.

Peptide-modified nanoparticles inhibit formation of Porphyromonas gingivalis biofilms with Streptococcus gordonii.

PurposeThe interaction of Porphyromonas gingivalis with commensal streptococci promotes P. gingivalis colonization of the oral cavity. We previously showed that a synthetic peptide (BAR) derived from Streptococcus gordonii potently inhibited the formation of P. gingivalis/S. gordonii biofilms (IC50 =1.3 µM) and reduced P. gingivalis virulence in a mouse model of periodontitis. Thus, BAR represents a novel therapeutic to control periodontitis by limiting P. gingivalis colonization of the oral cavity. Here, we sought to develop drug-delivery vehicles for potential use in the oral cavity that comprise BAR-modified poly(lactic-co-glycolic)acid (PLGA) nanoparticles (NPs).MethodsPLGA-NPs were initially modified with palmitylated avidin and subsequently conjugated with biotinylated BAR. The extent of BAR modification was quantified using a fluorescent-labeled peptide. Inhibition of P. gingivalis adherence to S. gordonii by BAR-modified NPs was compared with free peptide using a two-species biofilm model.ResultsBAR-modified NPs exhibited an average size of 99±29 nm and a more positive surface charge than unmodified NPs (zeta potentials of −7 mV and −25 mV, respectively). Binding saturation occurred when 37 nmol BAR/mg of avidin-NPs was used, which resulted in a payload of 7.42 nmol BAR/mg NPs. BAR-modified NPs bound to P. gingivalis in a dose-dependent manner and more potently inhibited P. gingivalis/S. gordonii adherence and biofilm formation relative to an equimolar amount of free peptide (IC50 of 0.2 µM versus 1.3 µM). BAR-modified NPs also disrupted the preformed P. gingivalis/S. gordonii biofilms more effectively than free peptide. Finally, we demonstrate that BAR-modified NPs promoted multivalent association with P. gingivalis, providing an explanation for the increased effectiveness of NPs.ConclusionThese results indicate that BAR-modified NPs deliver a higher local dose of peptide and may represent a more effective therapeutic approach to limit P. gingivalis colonization of the oral cavity compared to treatment with formulations of free peptide.

Description of a New Species ofHeterometrusEhrenberg, 1828 (Scorpiones: Scorpionidae) from Thailand with Remarks about the Utilization of Cytogenetic Data in Taxonomy of the Genus

A new species of the genus Heterometrus is described on the basis of a specimen recently collected in Thailand. Heterometrus minotaurus sp. nov. is morphologically closest to H. longimanus. The newly described species is well distinguished by its smaller overall size (83 mm) and shorter and less narrow metasoma with specific dorsolateral carinae on the fourth metasomal segment consisting of 9 or fewer granules. No females are known and so knowledge of sexual dimorphism in this species is currently lacking. In addition to the morphological characterization of H. minotaurus sp. nov., we present here also a description of the male holotype's karyotype. The diploid set of H. minotaurus sp. nov. consists of 54 chromosomes with a predominance of metacentrics, which gradually decrease in size. The presence of two types of multivalent association observed in postpachytene and metaphase I is commented on. Current knowledge of karyotypes of Heterometrus species is briefly summarized and compared with our cytogenetic results. In conclusion, we discuss the possible usefulness of karyotype as another interspecific feature applicable in the taxonomy of this scorpion group.

Microsporogenesis and meiotic abnormalities in different ‘Oblačinska’ sour cherry (Prunus cerasus L.) clones

The Oblačinska sour cherry (Prunus cerasus) is a mixture of different clones with similar tree and fruit characteristics that is indigenous to and widely planted in commercial orchards in Serbia. Sour cherry, including Oblačinska sour cherry clones, exhibits irregular meiosis which may contribute to low fruit set in some selections. The goal of this study was to examine the process of microsporogenesis and to determine if meiosis and its anomalies effect the in vitro pollen germination and pollen tube length in four ‘Oblačinska’ sour cherry clones that differ in fruit set and yields. All clones displayed varying degrees of chromosomal abnormalities in all meiosis sub-phases. The abnormalities became evident from late pachytene with more than half of the pollen mother cells (PMC) showing abnormal conjugation of chromosomes in metaphase I. The lowest number of PMCs with laggards was in clone III/9 and the highest in clone XIII/1. In second division, the univalent and multivalent association was observed at metaphase-II, the lagging and stickiness in anaphase-II, and the phenomenon persisted up to the microspore stage. In all four ‘Oblačinska’ clones, PMCs exhibited cytomixis phenomena, however, it was only observed in the second experimental year. Cytomixis differed among the four clones but was equally frequent in all stages of meiosis. The syncytia formed, most often consisted of 2–3 PMCs that were at the same phase of meiosis, and exhibited common cytoplasm and occasionally nuclear fusion. In vitro pollen germination and pollen tube length significantly differed between the clones. Most probably abnormalities during meiosis, regardless of the good results of pollen germination, influenced the reduced potential clones XI/3 and XIII/1.

Size dependence of steric shielding and multivalency effects for globular binding inhibitors.

Competitive binding inhibitors based on multivalent nanoparticles have shown great potential for preventing virus infections. However, general design principles of highly efficient inhibitors are lacking as the quantitative impact of factors such as virus concentration, inhibitor size, steric shielding, or multivalency effects in the inhibition process is not known. Based on two complementary experimental inhibition assays we determined size-dependent steric shielding and multivalency effects. This allowed us to adapt the Cheng-Prusoff equation for its application to multivalent systems. Our results show that the particle and volume normalized IC50 value of an inhibitor at very low virus concentration predominantly depends on its multivalent association constant, which itself exponentially increases with the inhibitor/virus contact area and ligand density. Compared to multivalency effects, the contribution of steric shielding to the IC50 values is only minor, and its impact is only noticeable if the multivalent dissociation constant is far below the virus concentration, which means if all inhibitors are bound to the virus. The dependence of the predominant effect, either steric shielding or multivalency, on the virus concentration has significant implications on the in vitro testing of competitive binding inhibitors and determines optimal inhibitor diameters for the efficient inhibition of viruses.

Militant Minority: British Columbia Workers and the Rise of a New Left, 1948–1972 by Benjamin Isitt (review)

Reviewed by: Militant Minority: British Columbia Workers and the Rise of a New Left, 1948–1972 by Benjamin Isitt Stuart Henderson Militant Minority: British Columbia Workers and the Rise of a New Left, 1948–1972 Benjamin Isitt Toronto: University of Toronto Press, 2011; 424 pages. $35.00 (paper), ISBN 9781442611054 To paraphrase an old saw, nobody disagrees with Marxists with more vehemence and conviction than other Marxists. This is one of the lessons of Canadian historian Benjamin Isitt’s Militant Minority, a dense and carefully presented study of working class politics in Canada’s westernmost province in a time of significant [End Page 169] upheaval. Tracing the political movements, battles and achievements on the British Columbian Left from the early Cold War period up to the triumphant (if unsustainable) victory of the New Democratic Party (NDP) in the provincial election of 1972, Militant Minority provides a thick and reasonably comprehensive approach to its complex subject. A deeply committed reading of the shift from an “Old” to a “New” Left politics (a shift that he, following American historian Maurice Isserman, takes pains to stress should not be understood as a rupture or a break but rather a continuity), Isitt offers a brave excursion into some pretty fraught territory. Indeed, this book aims in no uncertain terms to be provocative, and to unsettle its readers. Opening with an anecdote regarding a 1954 “incident” at which a representative of the Victoria Library Board declared that “any book which incites the downtrodden working man to revolt should be removed,” Isitt explains that he “hope[s] this book is worthy of that description” (3). And yet, as politically motivated as the author claims to be, Isitt is no mere cheerleader. The great strength of this book is, rather, in its unflinching determination to present a warts-and-all portrait of a multivalent (and not always pretty) association of left actors. Throughout this study Isitt is compelled to point out the inconsistencies, absurdities and stupidities that went hand in glove with the brilliance, commitment and integrity that characterized this burgeoning political movement. As a result, feathers will almost certainly be ruffled, as long-held beliefs are here frequently disassembled, and many names are named. Isitt explains that he has a three-fold purpose with Militant Minority: “(1) to restore political economy, and an emphasis on labour institutions, as valid subjects of working class and social history; (2) to provide a regional Canadian equivalent to Isserman’s study of the transition from the Old Left to the New Left, a ‘horizontal history’ grounded in the major political traditions of the Canadian working class and broader economic and political currents; and (3) to expand our understanding of B.C.’s political culture, class-polarized party system, and volatile labour-relations climate in a way that is relevant to a wider, interdisciplinary Canadian and international literature” (17). For the most part, Isitt succeeds on all three scores. [End Page 170] Beginning with a chapter devoted to “The Political Economy of British Columbia” is a clever decision, as the reader is provided with the wide and sturdy base onto which Isitt can build his arguments. (Indeed, for those readers unfamiliar with British Columbian politics in these years, or Canadian politics more generally, this chapter will prove utterly crucial to understanding the subsequent chapters—though broad in implication, Militant Minority is a highly local history.) This is followed by chapters on the fortunes of the British Columbia Communist Party (a.k.a. the Labour-Progressive party), the rise of the provincial CoOperative Commonwealth Federation (a social democratic party which would be reborn as the NDP in the 1960s), the outlying voices of Trotskyists and Maoists as well as anarchists and anti-war activists, the wildcat activism of (and within) many unions in the late 1960s, and a concluding chapter on the 1972 election which was won by the NDP. It must be said that Militant Minority is heavy on footnotes and appendices, even for an academic text—the book (based on a Ph.D. dissertation) appears to be twice as long as it actually is—and it is marked by an abundance of local detail and attention to internecine battles between...

Dendrimer-Based Multivalent Vancomycin Nanoplatform for Targeting the Drug-Resistant Bacterial Surface

Vancomycin represents the preferred ligand for bacteria-targeting nanosystems. However, it is inefficient for emerging vancomycin-resistant species because of its poor affinity to the reprogrammed cell wall structure. This study demonstrates the use of a multivalent strategy as an effective way for overcoming such an affinity limitation in bacteria targeting. We designed a series of fifth generation (G5) poly(amidoamine) (PAMAM) dendrimers tethered with vancomycin at the C-terminus at different valencies. We performed surface plasmon resonance (SPR) studies to determine their binding avidity to two cell wall models, each made with either a vancomycin-susceptible (D)-Ala-(D)-Ala or vancomycin-resistant (D)-Ala-(D)-Lac cell wall precursor. These conjugates showed remarkable enhancement in avidity in the cell wall models tested, including the vancomycin-resistant model, which had an increase in avidity of four to five orders of magnitude greater than free vancomycin. The tight adsorption of the conjugate to the model surface corresponded with its ability to bind vancomycin-susceptible Staphylococcus aureus bacterial cells in vitro as imaged by confocal fluorescent microscopy. This vancomycin platform was then used to fabricate the surface of iron oxide nanoparticles by coating them with the dendrimer conjugates, and the resulting dendrimer-covered magnetic nanoparticles were demonstrated to rapidly sequester bacterial cells. In summary, this article investigates the biophysical basis of the tight, multivalent association of dendrimer-based vancomycin conjugates to the bacterial cell wall, and proposes a potential new use of this nanoplatform in targeting Gram-positive bacteria.

Bivalent recognition of nucleosomes by the tandem PHD fingers of the CHD4 ATPase is required for CHD4-mediated repression

CHD4 is a catalytic subunit of the NuRD (nucleosome remodeling and deacetylase) complex essential in transcriptional regulation, chromatin assembly and DNA damage repair. CHD4 contains tandem plant homeodomain (PHD) fingers connected by a short linker, the biological function of which remains unclear. Here we explore the combinatorial action of the CHD4 PHD1/2 fingers and detail the molecular basis for their association with chromatin. We found that PHD1/2 targets nucleosomes in a multivalent manner, concomitantly engaging two histone H3 tails. This robust synergistic interaction displaces HP1γ from pericentric sites, inducing changes in chromatin structure and leading to the dispersion of the heterochromatic mark H3K9me3. We demonstrate that recognition of the histone H3 tails by the PHD fingers is required for repressive activity of the CHD4/NuRD complex. Together, our data elucidate the molecular mechanism of multivalent association of the PHD fingers with chromatin and reveal their critical role in the regulation of CHD4 functions.

Bivalent Recognition of a Single Nucleosome by the Tandem PHD Fingers of CHD4 is Required for CHD4-Mediated Repression

CHD4 is a catalytic subunit of the NuRD (nucleosome remodeling and deacetylase) complex essential in transcriptional regulation, chromatin assembly and DNA damage repair. CHD4 contains tandem plant homeodomain (PHD) fingers connected by a short linker, the biological function of which remains unclear. Here we explore the combinatorial action of the CHD4 PHD1/2 fingers and detail the molecular basis of their association with chromatin. We found that the inter-domain organization of PHD1/2 leads to specific targeting of a single nucleosome in a multivalent manner, concomitantly engaging both of the histone H3 tails. This robust synergistic interaction displaces HP1γ from pericentric sites, inducing changes in chromatin structure and dispersion of the heterochromatic marks H3K9me3 and H4K20me3. We demonstrate that recognition of the histone H3 tails by the PHD fingers is required for repressive activity of the CHD4/NuRD complex. Together, our data elucidate the molecular mechanism of multivalent association of the PHD fingers with chromatin and reveal their critical role in the regulation of CHD4 functions.

Interaction of Streptavidin-Based Peptide–MHC Oligomers (Tetramers) with Cell-Surface TCRs

The binding of oligomeric peptide-MHC (pMHC) complexes to cell surface TCR can be considered to approximate TCR-pMHC interactions at cell-cell interfaces. In this study, we analyzed the equilibrium binding of streptavidin-based pMHC oligomers (tetramers) and their dissociation kinetics from CD8(pos) T cells from 2C-TCR transgenic mice and from T cell hybridomas that expressed the 2C TCR or a high-affinity mutant (m33) of this TCR. Our results show that the tetramers did not come close to saturating cell-surface TCR (binding only 10-30% of cell-surface receptors), as is generally assumed in deriving affinity values (K(D)), in part because of dissociative losses from tetramer-stained cells. Guided by a kinetic model, the oligomer dissociation rate and equilibrium constants were seen to depend not only on monovalent association and dissociation rates (k(off) and k(on)), but also on a multivalent association rate (μ) and TCR cell-surface density. Our results suggest that dissociation rates could account for the recently described surprisingly high frequency of tetramer-negative, functionally competent T cells in some T cell responses.

A Double Plug–Socket System Capable of Molecular Keypad Locks through Controllable Photooxidation

Two robust divalent complexes have been successfully constructed by using complementary rigid spacers (anthracene vs. 1,4,5,8-naphthalenediimide (NDI)) and two pairs of [24]crown-8 ethers and secondary dialkylammonium functionalities as binding motifs. It was demonstrated that properly selected, rigid spacers are more efficient than flexible ones for achieving strong multivalent association. This is presumably due to the preorganization of the rigid spacers, the cooperation between charge-transfer interactions of rigid spacers, and the complexation of the binding motifs. Furthermore, the intermolecular photoinduced electron transfer (PET) between rigid spacers in these robust complexes could be switched on and off by modulating their complexation through acid-base reactions, which is reminiscent of a plug-socket system capable of electron transfer. In addition, the self-sensitized photooxidation of the divalent host with anthracene as a spacer can be completely inhibited after complexation with the divalent guests that contain NDI as spacers. This process could also be understood by invoking intermolecular PET and could be turned on and off through acid-base reactions. The photophysical and photochemical properties of these robust complexes have been interpreted as molecular keypad locks with alarm systems. Thus, a double plug-socket system and molecular keypad locks were successfully integrated inside robust multivalent systems and then the normal molecular devices were endowed with logic functions.

Cytogenetic Effects of Gamma Rays on Indica Rice Radha-4

The present study was conducted to understand the effects of different doses of radiations by gamma rays of Co60 on cytogenetics of indica rice, Radha-4 at the Central Lab, Institute of Agriculture and Animal Science, Rampur, Nepal. The immature PMC of M1 generation plants were analyzed performing the usual smearing and squashing technique. Radiation had a significant effect causing different types of cytogenetic aberrations, such as univalent and multivalent association of chromosomes, chromatin-bridges, laggards, fragments, stickiness, and multinucleated cells. An increasing number of aberrant cells with increasing dose of radiation was identified. The greatest share of aberration was identified due to multivalent association as a result of translocation followed by bridges and laggards, and the least effect for the stickiness of the chromosomes. Finally, 35 KR doses of gamma rays was the most potent dose inducing cytogenetic aberrations in meiotic pollen mother cell of rice compared to other dose spectrum.J. Inst. Agric. Anim. Sci. 27:25-36 (2006)

Synthesis of a fluorescent chitosan derivative and its application for the study of chitosan–mucin interactions

Fluorescein isothiocyanate-labeled chitosan was synthesized as a macromolecular fluorophore to investigate the molecular mechanism of interactions between chitosan and mucin by the fluorescence polarization method. The percent change in polarization upon chitosan–mucin association was measured as a function of the molecular weight of chitosan, and the pH and ionic strength of the medium. Dextran sulfate and albumin were used as positive and negative controls of polyelectrolyte complexation, respectively. The degree of polarization increased significantly with increasing mucin/chitosan (molecular weight, 750 000daltons) molar ratio in 0.10M acetic acid to suggest that there was multivalent binding between the two polymers. In contrast, only one dextran sulfate molecule was able to bind with the chitosan molecule. Albumin, having a net positive charge at a pH of 2.9, did not bind with chitosan. There was a possibility of multivalent association between high molecular weight chitosan and mucin as compared to univalent association with low molecular weight chitosan. The results of pH and ionic strength studies suggest that, in addition to electrostatic interactions between d-glucosamine residue of chitosan and the sialic acid residue of mucin, there could be other attractive forces involved in chitosan–mucin binding such as hydrogen bonding and/or hydrophobic interaction.

INVESTIGATION ON EMS AND DES INDUCED MEIOTIC ABERRATIONS IN CHILLI (Capsicum annum L.)

EMS and DES were found to be very potent chemical mutagens to induce the meiotic abnormalities in the chromosomes of Chilli (Capsicum annuum L.) var. G4. The meiotic abnormalities induced by mutagen were multivalent association, lagging chromosomes, stickiness of chromosomes. precocious movement of chromosomes, bridges, fragments and micronuclei etc. Multivalent association of chromosomes is due to reciprocal translocation in the chromosomes while bridges, laggards and precocious movement are attributed to the paracentric inversion, failure of chiasmata formation in the pairs and discrepancies is the spindle formation respectively. Chromosomal breakage is due to change in molecular constitutes of chromosomes whereas stickiness is result of depolymerisation of nucleic acid caused by mutagen.

Genetic variation in meiotic behaviour and fertility in tetraploid Hyoscyamus muticus: correlation with diploid meiosis

Autotetraploidy was induced in 19 morphologically distinct genotypes of the medicinal solanaceous plant Hyoscyamus muticus (Egyptian henbane, 4x= 56). The multivalent association frequencies at metaphase I of meiosis ranged from 0.328 to 0.628 and were positively correlated with the chiasmate association frequency in the diploid progenitors. The frequent absence of chiasmata in one of the two chromosome arms in most of the tetraploids was attributed to interference between the arms and to pairing partner exchange in one of the chromosome arms instead of around the centromers. The bivalent frequency in the tetraploids was positively correlated with pollen fertility and seed set. The latter ranged from 7.5 to 68.5 per cent. The asymmetry of the karyotype was not reflected in the distribution of open bivalents in the diploid. The regression coefficients of metaphase I chromosome associations over fertility in the tetraploid suggested that, in the range covered, one multivalent less resulted in an increase of about 12 per cent in seed set. The regression of open bivalent frequency in the diploid progenitor over seed set in the tetraploid suggested that one open bivalent more in the diploid would result in almost 18 per cent seed set in the tetraploid. It is concluded that the selection of diploid progenitors with high frequencies of open bivalents should enhance the fertility of the artificial polyploids in H. muticus.