Spatiotemporal Organization of AT- and GC-rich DNA and Their Association With Transition Proteins TP1 and TP2 in Rat Condensing Spermatids

Transition protein 1 (TP1) and TP2 replace histones during midspermiogenesis (stages 12-15) and are finally replaced by protamines. TPs play a predominant role in DNA condensation and chromatin remodeling during mammalian spermiogenesis. TP2 is a zinc metalloprotein with two novel zinc finger modules that condenses DNA in vitro in a GC-preference manner. TP2 also localizes to the nucleolus in transfected HeLa and Cos-7 cells, suggesting a GC-rich preference, even in vivo. We have now studied the localization pattern of TP2 in the rat spermatid nucleus. Colocalization studies using GC-selective DNA-binding dyes chromomycin A3 and 7-amino actinomycin D and an AT-selective dye, 4',6-diamidino-2-phenylindole, indicate that TP2 is preferentially localized to GC-rich sequences. Interestingly, as spermatids mature, TP2 and GC-rich DNA moves toward the nuclear periphery, and in the late stages of spermatid maturation, TP2 is predominantly localized at the nuclear periphery. Another interesting observation is the mutually exclusive localization of GC- and AT-rich DNA in the elongating and elongated spermatids. A combined immunofluorescence experiment with anti-TP2 and anti-TP1 antibodies revealed several foci of overlapping localization, indicating that TP1 and TP2 may have concerted functional roles during chromatin remodeling in mammalian spermiogenesis.

The Gerbera cDNA Microarray: A Tool for Large-Scale Identification of Genes Involved in Flower Development

During the past ten years, large-scale transcript analysis using microarrays has become a powerful tool to identify and predict functions for new genes. It allows simultaneous monitoring of the expression of thousands of genes and has become a routinely used tool in laboratories worldwide. Microarray analysis will, together with other functional genomics tools, take us closer to understanding the functions of all genes in genomes of living organisms. Flower development is a genetically regulated process which has mostly been studied in the traditional model species Arabidopsis thaliana, Antirrhinum majus and Petunia hybrida. The molecular mechanisms behind flower development in them are partly applicable in other plant systems. However, not all biological phenomena can be approached with just a few model systems. In order to understand and apply the knowledge to ecologically and economically important plants, other species also need to be studied. Sequencing of 17 000 ESTs from nine different cDNA libraries of the ornamental plant Gerbera hybrida made it possible to construct a cDNA microarray with 9000 probes. The probes of the microarray represent all different ESTs in the database. From the gerbera ESTs 20% were unique to gerbera while 373 were specific to the Asteraceae family of flowering plants. Gerbera has composite inflorescences with three different types of flowers that vary from each other morphologically. The marginal ray flowers are large, often pigmented and female, while the central disc flowers are smaller and more radially symmetrical perfect flowers. Intermediate trans flowers are similar to ray flowers but smaller in size. This feature together with the molecular tools applied to gerbera, make gerbera a unique system in comparison to the common model plants with only a single kind of flowers in their inflorescence. In the first part of this thesis, conditions for gerbera microarray analysis were optimised including experimental design, sample preparation and hybridization, as well as data analysis and verification. Moreover, in the first study, the flower and flower organ-specific genes were identified. After the reliability and reproducibility of the method were confirmed, the microarrays were utilized to investigate transcriptional differences between ray and disc flowers. This study revealed novel information about the morphological development as well as the transcriptional regulation of early stages of development in various flower types of gerbera. The most interesting finding was differential expression of MADS-box genes, suggesting the existence of flower type-specific regulatory complexes in the specification of different types of flowers. The gerbera microarray was further used to profile changes in expression during petal development. Gerbera ray flower petals are large, which makes them an ideal model to study organogenesis. Six different stages were compared and specifically analysed. Expression profiles of genes related to cell structure and growth implied that during stage two, cells divide, a process which is marked by expression of histones, cyclins and tubulins. Stage 4 was found to be a transition stage between cell division and expansion and by stage 6 cells had stopped division and instead underwent expansion. Interestingly, at the last analysed stage, stage 9, when cells did not grow any more, the highest number of upregulated genes was detected. The gerbera microarray is a fully-functioning tool for large-scale studies of flower development and correlation with real-time RT-PCR results show that it is also highly sensitive and reliable. Gene expression data presented here will be a source for gene expression mining or marker gene discovery in the future studies that will be performed in the Gerbera Laboratory. The publicly available data will also serve the plant research community world-wide.

Acetylation of Transition Protein 2 (TP2) by KAT3B (p300) Alters Its DNA Condensation Property and Interaction with Putative Histone Chaperone NPM3

The hallmark of mammalian spermiogenesis is the dramatic chromatin remodeling process wherein the nucleosomal histones are replaced by the transition proteins TP1, TP2, and TP4. Subsequently these transition proteins are replaced by the protamines P1 and P2. Hyperacetylation of histone H4 is linked to their replacement by transition proteins. Here we report that TP2 is acetylated in vivo as detected by anti-acetylated lysine antibody and mass spectrometric analysis. Further, recombinant TP2 is acetylated in vitro by acetyltransferase KAT3B (p300) more efficiently than by KAT2B (PCAF). In vivo p300 was demonstrated to acetylate TP2. p300 acetylates TP2 in its C-terminal domain, which is highly basic in nature and possesses chromatin-condensing properties. Mass spectrometric analysis showed that p300 acetylates four lysine residues in the C-terminal domain of TP2. Acetylation of TP2 by p300 leads to significant reduction in its DNA condensation property as studied by circular dichroism and atomic force microscopy analysis. TP2 also interacts with a putative histone chaperone, NPM3, wherein expression is elevated in haploid spermatids.Interestingly, acetylation of TP2 impedes its interaction with NPM3. Thus, acetylation of TP2 adds a new dimension to its role in the dynamic reorganization of chromatin during mammalian spermiogenesis.

DNA- and chromatin-condensing properties of rat testes H1a and H1t compared to those of rat liver H1bdec: H1t is a poor condenser of chromatin

Histones H1a and H1t are two major linker histone variants present at the pachytene interval of mammalian spermatogenesis. The DNA- and chromatin-condensing properties of these two variants isolated from rat testes were studied and compared with those from rat liver. For this purpose, the histone H1 subtypes were purified from the respective tissues using bath acid and salt extraction procedures, Circular dichroism studies revealed that acid exposure during isolation affects the alpha-helical structure of both the globular domain (in the presence of 1 M NaCl) and the C-terminal lambda-tail (in the presence of 60% trifluoroethanol). The condensation of rat oligonucleosomal DNA, as measured by circular dichroism spectroscopy, by the salt-extracted histone H1 was at least 10 times more efficient than condensation by the acid-extracted histone H1. A site size of 16-20 base pairs was calculated for the salt-extracted histone H1. Among the different histone H1 subtypes, somatic histone H1bdec had the highest DNA-condensing property, followed by histone H1a and histone H1t. All the salt-extracted histones condensed rat oligonucleosomal DNA more efficiently than linear pBR-322 DNA, Histones H1bdec and H1a condensed histone H1-depleted chromatin, prepared from rat liver nuclei, with relatively equal efficiency. On the other hand, there was no condensation of histone H1-depleted chromatin with the testes specific histone H1t. A comparison of the amino acid sequences of histone H1d (rat) and histone H1t (rat) revealed several interesting differences in the occurrence of DNA-binding motifs at the C-terminus. A striking observation is the presence of a direct repeat of an octapeptide motif K(A)T(S)PKKA(S)K(T)K(A) in histone H1d that is absent in histone H1t.

DNA-condensing and chromatin-condensing properties of rat testes hla and hit compared to those of rat-liver hlbdec - hlt is a poor condenser of chromatin

Histones H1a and H1t are two major linker histone variants present at the pachytene interval of mammalian spermatogenesis. The DNA- and chromatin-condensing properties of these two variants isolated from rat testes were studied and compared with those from rat liver. For this purpose, the histone H1 subtypes were purified from the respective tissues using bath acid and salt extraction procedures, Circular dichroism studies revealed that acid exposure during isolation affects the alpha-helical structure of both the globular domain (in the presence of 1 M NaCl) and the C-terminal lambda-tail (in the presence of 60% trifluoroethanol). The condensation of rat oligonucleosomal DNA, as measured by circular dichroism spectroscopy, by the salt-extracted histone H1 was at least 10 times more efficient than condensation by the acid-extracted histone H1. A site size of 16-20 base pairs was calculated for the salt-extracted histone H1. Among the different histone H1 subtypes, somatic histone H1bdec had the highest DNA-condensing property, followed by histone H1a and histone H1t. All the salt-extracted histones condensed rat oligonucleosomal DNA more efficiently than linear pBR-322 DNA, Histones H1bdec and H1a condensed histone H1-depleted chromatin, prepared from rat liver nuclei, with relatively equal efficiency. On the other hand, there was no condensation of histone H1-depleted chromatin with the testes specific histone H1t. A comparison of the amino acid sequences of histone H1d (rat) and histone H1t (rat) revealed several interesting differences in the occurrence of DNA-binding motifs at the C-terminus. A striking observation is the presence of a direct repeat of an octapeptide motif K(A)T(S)PKKA(S)K(T)K(A) in histone H1d that is absent in histone H1t.

A novel DNA intercalator, butylamino-pyrimido[4',5':4,5]selenolo(2,3-b)quinoline, induces cell cycle arrest and apoptosis in leukemic cells

DNA intercalators are one of the most commonly used chemotherapeutic agents. Novel intercalating compounds of pyrimido[4',5':4,5]selenolo(2,3-b)quinoline series having a butylamino or piperazino group at fourth position (BPSQ and PPSQ, respectively) are studied. Our results showed that BPSQ induced cytotoxicity whereas PPSQ was cytostatic. The cytotoxicity induced by BPSQ was concentration- and time-dependent. Cell cycle analysis and tritiated thymidine assay revealed that BPSQ affects the cell cycle progression by arresting at S phase. The absence of p-histone H3 and reduction in the levels of PCNA in the cells treated with BPSQ further confirmed the cell cycle arrest. Further, annexin V staining, DNA fragmentation, nuclear condensation and changes in the expression levels of BCL2/BAD confirmed the activation of apoptosis. Activation of caspase 8 and lack of cleavage of caspase 9, caspase 3 and PARP suggest the possibility of BPSQ triggering extrinsic pathway for induction of apoptosis, which is discussed. Hence, we have identified a novel compound which would have clinical relevance in cancer chemotherapeutics.

Specific Small-Molecule Activator of Aurora Kinase A Induces Autophosphorylation in a Cell-Free System

Aurora kinases are essential for chromosomal segregation and cell division and thereby important for maintaining the proper genomic integrity. There are three classes of aurora kinases in humans: A, B, and C. Aurora kinase A is frequently overexpressed in various cancers. The link of the overexpression and tumorigenesis is yet to be understood. By employing virtual screening, we have found that anacardic acid, a pentadecane aliphatic chain containing hydroxylcarboxylic acid, from cashew nut shell liquid could be docked in Aurora kinases A and B. Remarkably, we found that anacardic acid could potently activate the Aurora kinase A mediated phosphorylation of histone H3, but at a similar concentration the activity of aurora kinase B remained unaffected in vitro. Mechanistically, anacardic acid induces the structural changes and also the autophosphorylation of the aurora kinase A to enhance the enzyme activity. This data thus indicate anacardic acid as the first small-molecule activator of Aurora kinase, which could be highly useful for probing the function of hyperactive (overexpressed) Aurora kinase A.

Transcription of Human Resistin Gene Involves an Interaction of Sp1 with Peroxisome Proliferator-Activating Receptor Gamma (PPAR gamma)

Background: Resistin is a cysteine rich protein, mainly expressed and secreted by circulating human mononuclear cells. While several factors responsible for transcription of mouse resistin gene have been identified, not much is known about the factors responsible for the differential expression of human resistin.Methodology/Principal Finding: We show that the minimal promoter of human resistin lies within similar to 80 bp sequence upstream of the transcriptional start site (-240) whereas binding sites for cRel, CCAAT enhancer binding protein alpha (C/EBP-alpha), activating transcription factor 2 (ATF-2) and activator protein 1 (AP-1) transcription factors, important for induced expression, are present within sequences up to -619. Specificity Protein 1(Sp1) binding site (-276 to -295) is also present and an interaction of Sp1 with peroxisome proliferator activating receptor gamma (PPAR gamma) is necessary for constitutive expression in U937 cells. Indeed co-immunoprecipitation assay demonstrated a direct physical interaction of Sp1 with PPAR gamma in whole cell extracts of U937 cells. Phorbol myristate acetate (PMA) upregulated the expression of resistin mRNA in U937 cells by increasing the recruitment of Sp1, ATF-2 and PPAR gamma on the resistin gene promoter. Furthermore, PMA stimulation of U937 cells resulted in the disruption of Sp1 and PPAR gamma interaction. Chromatin immunoprecipitation (ChIP) assay confirmed the recruitment of transcription factors phospho ATF-2, Sp1, Sp3, PPAR gamma, chromatin modifier histone deacetylase 1 (HDAC1) and the acetylated form of histone H3 but not cRel, C/EBP-alpha and phospho c-Jun during resistingene transcription.Conclusion: Our findings suggest a complex interplay of Sp1 and PPAR gamma along with other transcription factors that drives the expression of resistin in human monocytic U937 cells.

Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge

Influenza HA is the primary target of neutralizing antibodies during infection, and its sequence undergoes genetic drift and shift in response to immune pressure. The receptor binding HA1 subunit of HA shows much higher sequence variability relative to the metastable, fusion-active HA2 subunit, presumably because neutralizing antibodies are primarily targeted against the former in natural infection. We have designed an HA2-based immunogen using a protein minimization approach that incorporates designed mutations to destabilize the low pH conformation of HA2. The resulting construct (HA6) was expressed in Escherichia coli and refolded from inclusion bodies. Biophysical studies and mutational analysis of the protein indicate that it is folded into the desired neutral pH conformation competent to bind the broadly neutralizing HA2 directed monoclonal 12D1, not the low pH conformation observed in previous studies. HA6 was highly immunogenic in mice and the mice were protected against lethal challenge by the homologous A/HK/68 mouse-adapted virus. An HA6-like construct from another H3 strain (A/Phil/2/82) also protected mice against A/HK/68 challenge. Regions included in HA6 are highly conserved within a subtype and are fairly well conserved within a clade. Targeting the highly conserved HA2 subunit with a bacterially produced immunogen is a vaccine strategy that may aid in pandemic preparedness.

Mode of action of antitumour antibiotics-III: Modulation of permeability of nuclear membrane in the presence of the antibiotics

The binding characteristics of the antibiotics to nuclei and their effect on the permeability of nuclear membrane with respect to histones and ribonucleic acids have been investigated. The binding constant for chromomycin A3 was found to be 1.4 × 104M?1 and number of binding sites was equal to 3.48 ± 1.08 × 1012 molecules/nuclei. The antibiotic chromomycin A3 enhanced the uptake of lysine-rich histone, actinomycin D decreased the uptake and ethidium bromide had no effect. Chromomycin A3 also enhanced the release of acid insoluble fraction containing RNA from the nuclei, actinomycin D and ethidium bromide inhibited the release of acid insoluble fraction containing RNA. The relevance of this finding to the role of nuclear envelope in understanding the mechanism of action of the antibiotic has been discussed.

Nucleosomes on linear duplex DNA allow homologous pairing but prevent strand exchange promoted by RecA protein

To understand the molecular basis of gene targeting, we have studied interactions of nucleoprotein filaments comprised of single-stranded DNA and RecA protein with chromatin templates reconstituted from linear duplex DNA and histones. We observed that for the chromatin templates with histone/DNA mass ratios of 0.8 and 1.6, the efficiency of homologous pairing was indistinguishable from that of naked duplex DNA but strand exchange was repressed. In contrast, the chromatin templates with a histone/DNA mass ratio of 9.0 supported neither homologous pairing nor strand exchange. The addition of histone H1, in stoichiometric amounts, to chromatin templates quells homologous pairing. The pairing of chromatin templates with nucleoprotein filaments of RecA protein-single-stranded DNA proceeded without the production of detectable networks of DNA, suggesting that coaggregates are unlikely to be the intermediates in homologous pairing. The application of these observations to strategies for gene targeting and their implications for models of genetic recombination are discussed.

Preparation and characterization of hydrazine derivatives. Part-II: Reaction of transition metal ammonium double sulphates with hydrazine hydrate

Transition metal ammonium double sulphates (NH4)2M(SO4)2· 6H2O, where M = Fe, Co and Ni react with hydrazine hydrate in air giving crystalline compounds of the general formula (N2H5) [M(N2H3COO)3] H2O. The reaction proceeds through (N2H5)2 M(SO4)2, · 3N2H4, (N2H5)2 [M(OH)4 · (N2H4)2], M(N2H3COO)2 · (N2H4)2 and N2H5 [M(N2 H3 COO)3] intermediates. The reaction sequence is followed by chemical analysis and infrared spectra. A possible reaction mechanism has been suggested.

Syntheses, spectroscopy, structures, and conformations of .lambda.3-cyclotriphosphazanes: role of negative hyperconjugation

The reactions of As-chlorocyclotriphosphazane [EtNPCl], with phenols or trifluoroethanol yield the respective aryloxy- or trifluoroethoxy-containingX 3-cyclotriphosphazanes [EtNP(OR)]3 (R = C6H4Br-4 (2),C 6H5 (3C,6 H3-Mez-3,5 (4), C6H3Mez-2,6 (5), CH2CF3 (6)) as their cis-transisomericmixtures. The products have beencharacterized by IRand NMRspectroscopy. Thecrystalstructuresofboth thecis (2a) and trans(2b) isomer_softhep-bromophenoxy derivative have been determined by X-ray diffraction. Crystal data for 2a: triclinic, P1, a = 9.872(4) A, b = 13.438(6) A, c = 13.548(8) A, CY = 117.02(5)', 0 = 96.00(6)', y = 105.38(4)O, Z = 2, final R = 0.080. Crystal data for 2b: monoclinic, P21/n, a = 12.721(6) A, b = 13.468(7) A, c = 17.882(5) A, /3 = 101.62(3)O, Z = 4, final R = 0.066. The cis isomer exhibits a chair-triaxial conformation and the trans isomer a boat-triaxial conformation. Conformational preferences of X3-cyclotriphosphazanes have been probed by both MNDO and ab initio calculations on model systems [HNPXIp (X = H, F). In addition to vicinal lone pair repulsions, negative hyperconjugative interactions involving the nitrogen lone pairs and adjacent P-X Q* orbitals are found to be important (especially when X is an electronegative substituent) in determining the conformational preferences of X3-cyclotriphosphazanes. The calculations also show that the axial - equatorial conversion at phosphorus has a large activation barrier in these systems

Testis-specific histone (H1t) is not phosphorylated and has a weak interaction with chromatin

Soluble chromatin was prepared from rat testes after a brief micrococcal nuclease digestion. After adsorption onto hydroxylapatite at low ionic strength, the histone Hl subtypes were eluted with a shallow salt gradient of 0.3 M NaCl to 0.7 M NaCl. Histone Hlt was eluted at 0.4 MNaCl, while histones H1a and Hlc were eluted at 0.43 M NaCl and 0.45 M respectively. The extreme divergence of the amino acid sequence of the C-terminal half of histone Hlt, the major DNA binding domain of histone Hl, from that of the somatic consensus sequence may contribute to the weaker interaction of histone Hlt with the rat testis chromatin. Further, histone Hlt was not phosphorylated in vivo in contrast to histone Hla and Hlc, as is evident from the observation that histone Hlt lacks the SPKK motif recognized by the CDC-2kinase or the RR/KXS motif recognized by protein kinase A.

Fourier transform infrared microspectroscopy identifies protein propionylation in histone deacetylase inhibitor treated glioma cells

Histone deacetylase inhibitors (HDIs) have attracted considerable attention as potential drug molecules in tumour biology. In order to optimise chemotherapy, it is important to understand the mechanisms of regulation of histone deacetylase (HDAC) enzymes and modifications brought by various HDIs. In the present study, we have employed Fourier transform infrared microspectroscopy (FT-IRMS) to evaluate modifications in cellular macromolecules subsequent to treatment with various HDIs. In addition to CH3 (methyl) stretching bands at 2872 and 2960 cm1, which arises due to acetylation, we also found major changes in bands at 2851 and 2922 cm1, which originates from stretching vibrations of CH2 (methylene) groups, in valproic acid treated cells. We further demonstrate that the changes in CH2 stretching are concentration-dependent and also induced by several other HDIs. Recently, HDIs have been shown to induce propionylation besides acetylation [1]. Since propionylation involves CH2 groups, we hypothesized that CH2 vibrational frequency changes seen in HDI treated cells could arise due to propionylation. As verification, pre-treatment of cells with propionyl CoA synthetase inhibitor resulted in loss of CH2 vibrational changes in histones, purified from valproic acid treated cells. This was further proved by western blot using propionyl-lysine specific antibody. Thus we demonstrate for the first time that propionylation could be monitored by studying CH2 stretching using IR spectroscopy and further provide a platform for monitoring HDI induced multiple changes in cells. (C) 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)