Crystal structures, dynamics and functional implications of molybdenum-cofactor biosynthesis protein MogA from two thermophilic organisms

Molybdenum-cofactor (Moco) biosynthesis is an evolutionarily conserved pathway in almost all kingdoms of life, including humans. Two proteins, MogA and MoeA, catalyze the last step of this pathway in bacteria, whereas a single two-domain protein carries out catalysis in eukaryotes. Here, three crystal structures of the Moco-biosynthesis protein MogA from the two thermophilic organisms Thermus thermophilus (TtMogA; 1.64 angstrom resolution, space group P2(1)) and Aquifex aeolicus (AaMogA; 1.70 angstrom resolution, space group P2(1) and 1.90 angstrom resolution, space group P1) have been determined. The functional roles and the residues involved in oligomerization of the protein molecules have been identified based on a comparative analysis of these structures with those of homologous proteins. Furthermore, functional roles have been proposed for the N- and C-terminal residues. In addition, a possible protein-protein complex of MogA and MoeA has been proposed and the residues involved in protein-protein interactions are discussed. Several invariant water molecules and those present at the subunit interfaces have been identified and their possible structural and/or functional roles are described in brief. In addition, molecular-dynamics and docking studies with several small molecules (including the substrate and the product) have been carried out in order to estimate their binding affinities towards AaMogA and TtMogA. The results obtained are further compared with those obtained for homologous eukaryotic proteins.

Quantitation of spermatogenesis by DNA flow cytometry: Comparative study among six species of mammals

Suspensions of testicular germ cells from six species of mammals were prepared and stained for the DNA content with a fluorochrome (ethidium bromide) adopting a common technique and subjected to DNA flow cytometry. While uniform staining of the germ cells of the mouse, hamster, rat and monkey could be obtained by treating with 0.5% pepsin for 60 min followed by staining with ethidium bromide for 30 min, that of the guinea pig and rabbit required for optimal staining pepsinization for 90 min and treatment with ethidium bromide for 60 min. The procedure adopted here provided a uniform recovery of over 80% of germ cells with each one of the species tested and the cell population distributed itself according to the DNA content (expressed as C values) into 5 major classes-spermatogonia (2C), cells in S-phase, primary spermatocytes (4C), round spermatids (1C), and elongating/elongated spermatids (HC). Comparison of the DNA distribution pattern of the germ cell populations between species revealed little variation in the relative quantities of cells with 2C (8-11%), S-phase (6-9%), and 4C (6-9%) amount of DNA. Though the spermatid cell populations exhibited variations (1C:31-46%, HCI:7-20% and and HC2:11-25%) they represented the bulk of germ cells (70-80%). The overall conversion of 2C to 1C (1C:2C ratio) and meiotic transformation of 4C cells to IC (1C:4C ratio) kinetics were relatively constant between the species studied. The present study clearly demonstrates that DNA flow cytometry can be adopted with ease and assurance to quantify germ cell transformation and as such spermatogenesis by analysing a large number of samples with consistency both within and across the species barrier. Any variation from the norms in germ cell proportions observed following treatment, for e.g. hormonal stimulation or deprivation can then be ascribed due to a specific effect of the hormone/drug on single/multiple steps in germ cell transformation

Development of specific DNA probes and their usage in the detection of Plasmodium vivax infection in blood

The application of nucleic acid probes, in the detection of pathogenic micro-organisms, has become an integral part of diagnostic technologies. In this study, Plasmodium vivax-specific DNA probes have been identified by carrying out genomic subtractive hybridization. In this approach, the recombinant clones from a P. vivax genomic library are screened with radiolabelled human and P. falciparum DNA. The colonies which react with labelled P. falciparum and human DNA are eliminated and those which do not produce any autoradiographic signal have been subjected to further screening procedures. Three Fl vivax specific DNA probes have been obtained by these repeated screenings. Further analyses indicate that these probes are specific and sensitive enough to detect P. vivax infection in clinical blood samples when used in a non-radioactive DNA hybridization assay. (C) 1995 Academic Press Limited

Photocytotoxicity and DNA photocleavage activity of La(III) and Gd(III) complexes of phenanthroline bases

Lanthanide(II) complexes La(B)(acac)(3)] (1-3) and Gd(B)(acac)(3)] (4-6), where B is a N,N-donor phenanthroline base, viz., 1,10-phenanthroline (phen in 1, 4), dipyrido3,2-d:2',3'-f]quinoxaline (dpq in 2, 5) and dipyrido3,2-a:2',3'-c]phenazine (dppz in 3, 6), have been prepared and characterized. The Gd(111) complexes 4 6 are structurally characterized by single crystal X-ray crystallography. The complexes display GdO6N2 coordination with the ligands showing bidentate chelating mode of bonding. The complexes are non-electrolytic in aqueous DMF and exhibit ligand-centered absorption bands in the UV region. The dppz complexes show a band at 380 nm in DMF. The La(111) complexes are diamagnetic. The Gd(III) complexes are paramagnetic with magnetic moment that corresponds to seven unpaired electrons. The Complexes are avid binders to calf thymus DNA giving K-b values in the range of 4.7 x 10(4) 6.1 x 10(5) M-1 with a relative binding order: 3, 6 (dppz) > 2, 5 (dpq) > 1, 4 (phen). The binding data suggest DNA surface and/or groove binding nature of the complexes. The dpq and dppz complexes efficiently cleave SC DNA to its nicked circular form in UV-A light of 365 nm via formation of both singlet oxygen (O-1(2)) and hydroxyl radical (HO center dot) species. The dppz complexes 3 and 6 exhibit significant PDT effect in He La cervical cancer cells giving respective IC50 value of 460(+/- 50) and 530(+/- 30) nM in UV-A light of 365 rim, and are essentially non-toxic in dark with an IC50 value of >100 mu M. The dppz ligand alone is cytotoxic in dark and UV-A light. A significant decrease in the dark toxicity of the dppz base is observed on binding to the Ln(III) ion while retaining its photocytotoxicity.

Unwinding of heterologous DNA by RecA protein during the search for homologous sequences

The search for homologous sequences promoted by RecA protein in vitro involves a presynaptic filament and naked duplex DNA, the multiple contacts of which produce nucleoprotein networks or coaggregates. The single-stranded DNA within the presynaptic filaments, however, is extended to an axial spacing 1.5 times that of B-form DNA. To investigate this paradoxical difference between the spacing of bases in the RecA presynaptic filament versus the target duplex DNA, we explored the effect of heterologous contacts on the conformation of DNA, and vice versa. In the presence of wheat germ topoisomerase I, RecA presynaptic filaments induced a rapid, limited reduction in the linking number of heterologous circular duplex DNA. This limited unwinding of heterologous duplex DNA, termed heterologous unwinding, was detected within 30 seconds and reached a steady state within a few minutes. Presynaptic filaments that were formed in the presence of ATP?S and separated from free RecA protein by gel filtration also generated a ladder of topoisomers upon incubation with relaxed duplex DNA and topoisomerase. The inhibition of heterologous contacts by 60 mImage -NaCl or 5 mImage -ADP resulted in a corresponding decrease in heterologous unwinding. In reciprocal fashion, the stability or number of heterologous contacts with presynaptic filaments was inversely related to the linking number of circular duplex DNA. These observations show that heterologous contacts with the presynaptic filament cause a limited unwinding of the duplex DNA, and conversely that the ability of the DNA to unwind stabilizes transient heterologous contacts.

DNA Condensation by the Rat Spermatidal Protein TP2 Shows GC-Rich Sequence Preference and Is Zinc Dependent

Transition protein-2 (TP2), isolated from rat testes, was recently shown to be a zinc metalloprotein. We have now carried out a detailed analysis of the DNA condensing properties of TP2 with various polynucleotides using circular dichroism spectroscopy. The condensation of the alternating copolymers by TP2 (incubated with 10 mu M ZnSO4), namely, poly(dG-dC). poly(dG-dC) and poly(dA-dT). poly(dA-dT), was severalfold higher than condensation of either of the homoduplexes poly(dG). poly-(dC) and poly(dA). poly(dT) or rat oligonucleosomal DNA. Between the two alternating copolymers, poly(dG-dC). poly(dG-dC) was condensed 3.2-fold more effectively than poly(dA-dT). poly(dA-dT). Preincubation of TP2 with 5 mM EDTA significantly reduced its DNA-condensing property. Interestingly, condensation of the alternating copolymer poly(dI-dC). poly(dI-dC) by TP2 was much less as compared to that of poly(dG-dC). poly(dG-dC). The V8 protease-derived N-terminal fragment (88 aa) condensed poly(dA-dT). poly(dA-dT) to a very small extent but did not have any effect on poly(dG-dC). poly-(dG-dC). The C-terminal fragment (28 aa) was able to condense poly(dA-dT) . poly(dA-dT) more effectively than poly(dG-dC). poly(dG-dC). These results suggest that TP2 in its zinc-coordinated form condenses GC-rich polynucleotides much more effectively than other types of polynucleotides. Neither the N-terminal two-thirds of TP2 which is the zinc-binding domain nor the C-terminal basic domain are as effective as intact TP2 in bringing about condensation of DNA.

DNA recognition by the EcoP15I and EcoPI modification methyltransferases

The DNA-binding properties of the EcoP15I DNA methyltransferase (M . EcoP15I; MTase) were studied using electrophoretic mobility shift assays. We show by molecular size-exclusion chromatography and dimethyl suberimidate crosslinking that M . EcoP15I is a dimer in solution. While M . EcoP15I binds approx. threefold more tightly to its recognition sequence, 5'-CAGCAG-3', than to non-specific sequences in the presence of AdoMet or its analogs, the discrimination between specific and non-specific sequences significantly increases in presence of ATP. These results suggest for the first time a role for ATP in DNA recognition by type-III restriction-modification enzymes. Furthermore, we show that although c2 EcoPI mutant MTases are defective in AdoMet binding, they are still able to bind DNA in a sequence-specific manner.

Isolation of an 800 kb contiguous DNA fragment encompassing a 3.5-cM region of chromosome 1 in Arabidopsis using YAC clones

The apetalal mutation of Arabidopsis affects floral meristem identity and the development of sepal and petal primordia of the flower. We mapped the available RFLP markers on chromosome 1 that are in the general vicinity of apetalal on a fine structure map and then chose the closest RFLP as a starting point for contiguous DNA (contig) generation. We report here a contig of about 800 kilobases (kb) that spans a 3.5 cM region of chromosome 1. We used genomic libraries of Arabidopsis prepared in yeast artificial chromosome (YAC) vectors and the detailed characterization of 19 YACs is reported. RFLPs displayed by the end fragments from the walk were mapped to align and correlate the genetic and physical maps for this region of chromosome 1. In this segment of the genome, 1 cM corresponds to a little over 200 kb of physical distance.

DNA binding properties of some cationic acridine derivatives

Four cationic acridine derivatives have been synthesized. The positively charged amine residue in one of these is connected directly on to the acridine nucleus and in three other acridines, the amines are connected via a 9-CH2 unit to acridine. We have investigated the binding of these acridines with mammalian DNA by absorption titration, UV- and induced-CD spectroscopy and competitive ethidium bromide displacement fluorescence assay. The effects on the DNA duplex denaturation melting temperatures upon binding of each one of these are also examined. The results obtained herein clearly show that the introduction of a -CH2 group in the im mediate vicinity of the interrelation moiety introduces alterations in the DNA binding characteristics of the resulting acridines.

Purification and characterization of DNA-dependent RNA polymerase II from Candida utilis

DNA-dependent RNA polymerase II from Candida utilis has been purified to near homogeneity. The purified enzyme resolved into three subforms, viz. IIO, IIA and IIB. On SDS-PAGE the enzyme showed ten polypeptides with molecular weights in the range of 205 kDa to 14 kDa. By two dimensional electrophoresis (IEF followed by SDS-PAGE) the presence of basic and acidic polypeptides has been demonstrated. The enzyme showed Km values of 5, 5.6 and 8 mu M for GTP, CTP and ATP, respectively, and the activity was inhibited by low levels of oc-amanitin and antibodies raised against bovine RNA polymerase II. By Western blot analysis the enzyme was found to cross-react with antibodies to bovine RNA polymerase II. RNA polymerase II from G. utilis is a phosphoprotein, the subunits RPB1 and RPB10 were found to be phosphorylated. Analysis of carboxy-terminal domain indicated that it was functionally redundant at least in case of nonspecific transcription, implicating its role in other nuclear processes, such as promoter specific initiation or transcription activation or RNA processing.

Schiff base oxovanadium(IV) complexes of phenanthroline bases showing DNA photocleavage activity at near-IR light and photocytotoxicity

Oxovanadium(IV) complexes VO(L)(B)](ClO4) (1-3) of N-2-pyridylmethylidine-2-hydroxyphenylamine (HL) Schiff base and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyrido3,2-d: 2',3'-f] quinoxaline (dpq in 2) or dipyrido3,2-a: 2',3'-c] phenazine (dppz in 3), were prepared, characterized and their DNA binding property, photo-induced DNA cleavage activity and photocytotoxicity in HeLa cells studied. The crystal structure of 1 shows the presence of a VO2+ moiety in VO2N4 coordination geometry. The complexes show a d-d band at similar to 830 nm in DMF. The complexes display an oxidative V(V)-V(IV) response near 0.5 V versus SCE and a reductive V(IV)/V(III) response near -0.65 V in DMF -0.1 M TBAP. The complexes that are avid binders to CT DNA giving K-b values within 7.1 x 10(4) to 3.2 x 10(5) M-1, do not show any significant chemical nuclease activity in presence of 3-mercaptopropionic acid or glutathione. The dpq and dppz complexes are photocleavers of pUC19 DNA in UV-A light of 365 nm forming both O-1(2) and (OH)-O-center dot radicals and in near-IR light of 785 nm forming (OH)-O-center dot radicals. The dppz complex exhibits photocytotoxicity in visible light in HeLa cells (IC50 = 6.8 mu M). Flow-cytometric study on this complex shows a high sub-G1 phase in light compared to dark indicating PDT effect. (C) 2011 Elsevier B. V. All rights reserved.

Preferential condensation of SAR-DNA by histone H1 and its SPKK containing octapeptide repeat motif

Linker histone H1 binds preferentially the scaffold associated region (SAR) DNA elements that contain characteristic oligo dA . dT tracts. In the present study, we have compared the condensation brought about by histone H1 of a SAR DNA fragment in the histone spacer region of Drosophila melanogaster with that of a random DNA (pBR322 EcoRI-SalI) fragment by circular dichroism spectroscopy. The condensation of the SAR DNA fragment by histone H1 is 3-4-fold higher than that of the random DNA fragment. A 16-mer peptide, ATPKKSTKKTPKKAKK, the sequence that is present in the C-terminus of histone H1d, which has recently been shown to possess DIVA and chromatin condensing properties, also condenses the SAR DNA fragment preferentially in a highly cooperative manner. We have proposed a model for the dynamics of chromatin structure involving histone H1-SAR DNA interaction through SPKK containing peptide motifs and its competition by AT-hook peptides present in the nonhistone chromosomal proteins like HMG-I and HMG-Y.

Regulation of progesterone biosynthesis in the human placenta by estradiol 17 beta and progesterone

Ex vivo addition of estradiol 17 beta to first trimester or term human placental minces caused a significant increase in the quantity of progesterone produced. Addition of an aromatase inhibitor, CGS 16949 A or the estrogen receptor antagonist, ICI 182780, significantly inhibited progesterone production confirming the role of estradiol 17 beta in the regulation of progesterone synthesis in human placenta. RU 486 and ZK 98299, which are antagonists of progesterone receptor, significantly modulated progesterone synthesis in the human placenta but exhibited paradoxical effects on the first trimester and term placenta We conclude that progesterone synthesis in the human placenta is regulated by estradiol 17 beta and progesterone. This is the first report providing evidence for autoregulation of progesterone synthesis in the human placenta.

Role of hydrophobic effect and surface charge in surfactant-DNA association

Ethidium bromide is one of the best known DNA intercalator. Upon intercalation inside DNA, the fluorescence due to ethidium bromide gets enhanced by many orders of magnitude. In this paper, we employed ethidium bromide as a probe for studying surfactant-DNA complexation using fluorescence spectroscopy and agarose gel electrophoresis. Surfactants of different charge types and chain lengths were used and the results were compared with that of the related small organic cations or salts under comparable conditions. The cationic surfactants induced destabilization of the ethidium bromide-DNA complex at concentrations in orders of magnitude lower than that of the small organic cations or salts. In contrast however, the anionic surfactants failed to promote any such destabilization of probe-DNA complex. DNA loses its ethidium bromide stainability in the presence of high concentration of cationic surfactant aggregates as revealed from agarose gel electrophoresis experiments. Inclusion of surfactants and other additives into the DNA generally enhanced the DNA double-strand to single strand transition melting temperatures by a few degrees, in a concentration-dependent manner and at high surfactant concentration melting profiles got broadened.