Interaction of palladium (II) with DNA

The nature of interaction of palladium(II) with calf thymus DNA was studied using viscometry, ultraviolet, visible and infrared spectrophotometry and optical rotatory disperison and circular dichroism measurements. The results indicate that Pd(II) interacts with both the phosphate and bases of DNA. The ORD/CD data indicate that the binding of Pd(II) to DNA brings about considerable conformational changes in DNA.

Interaction of DNA with anti-cancer drugs: copper-thiosemicarbazide system

The interaction of copper-thiosemicarbazide complexes with DNA was investigated using ultraviolet and infrared spectroscopy. Evidence for the interaction of the complexes with nucleic acid bases and with the phosphate group is presented.

New ternary copper(II) complexes of L-alanine and heterocyclic bases: DNA binding and oxidative DNA cleavage activity

Four new ternary copper(II) complexes of alpha-amino acid having polypyridyl bases of general formulation [Cu(L-ala)(B)(H2O)](X)(1-4), where L-ala is L-alanine, B is an N,N-donor heterocyclic base, viz. 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2) and 5,6-phenanthroline dione (dione, 3), dipyrido[3,2:2',3'-f] quinoxaline (dpq, 4), and X = ClO4-/NO3- are synthesized, characterized by various spectroscopic and X-ray crystallographic methods. The complexes show a distorted square-pyramidal (4 + 1) CuN3O2 coordination geometry. The one-electron paramagnetic complexes (1-4) display a low energy d-d band near 600 nm in aqueous medium and show a quasi-reversible cyclic voltammetric response due to one-electron Cu(II)/Cu(I) reduction near - 100 mV (versus SCE) in DMF-0.1 M TBAP. Binding interactions of the complexes with calf thymus DNA (CT-DNA) were investigated by UV-Vis absorption titration, ethidium bromide displacement assay, viscometric titration experiment and DNA melting studies. All the complexes barring the complexes 1 and 3 are avid binder to the CT-DNA in the DNA minor groove giving an order: 4 > 2 >>>1, 3. The complexes 2 and 4 show appreciable chemical nuclease activity in the presence of 3-mercaptopropionic acid (MPA) as a reducing agent. Hydroxyl radical was investigated to be the DNA cleavage active species. Control experiments in the presence of distamycin-A show primarily minor groove-binding propensity for the complexes 2 and 4 to the DNA.

Archaea, Bacteria, and methane production along environmental gradients in fens and bogs

Metanogeenit ovat hapettomissa oloissa eläviä arkkien pääryhmään kuuluvia mikrobeja, joiden ainutlaatuisen aineenvaihdunnan seurauksena syntyy metaania. Ilmakehässä metaani on voimakas kasvihuonekaasu. Yksi suurimmista luonnon metaanilähteistä ovat kosteikot. Pohjoisten soiden metaanipäästöt vaihtelevat voimakkaasti eri soiden välillä ja yhden suon sisälläkin, riippuen muun muassa vuodenajasta, suotyypistä ja kasvillisuudesta. Väitöskirjatyössä tutkittiin metaanipäästöjen vaihtelun mikrobiologista taustaa. Tutkimuksessa selvitettiin suotyypin, vuodenajan, tuhkalannoituksen ja turvesyvyyden vaikutusta metanogeeniyhteisöihin sekä metaanintuottoon kolmella suomalaisella suolla. Lisäksi tutkittiin ei-metanogeenisia arkkeja ja bakteereita, koska ne muodostavat metaanin tuoton lähtöaineet osana hapetonta hajotusta. Mikrobiyhteisöt analysoitiin DNA- ja RNA-lähtöisillä, polymeraasiketjureaktioon (PCR) perustuvilla menetelmillä. Merkkigeeneinä käytettiin metaanin tuottoon liittyvää mcrA-geeniä sekä arkkien ja bakteerien ribosomaalista 16S RNA-geeniä. Metanogeeniyhteisöt ja metaanintuotto erosivat huomattavasti happaman ja vähäravinteisen rahkasuon sekä ravinteikkaampien sarasoiden välillä. Rahkasuolta löytyi lähes yksinomaan Methanomicrobiales-lahkon metanogeeneja, jotka tuottavat metaania vedystä ja hiilidioksidista. Sarasoiden metanogeeniyhteisöt olivat monimuotoisempia, ja niillä esiintyi myös asetaattia käyttäviä metanogeeneja. Vuodenaika vaikutti merkittävästi metaanintuottoon. Talvella havaittiin odottamattoman suuri metaanintuottopotentiaali sekä viitteitä aktiivisista metanogeeneista. Arkkiyhteisön koostumus sen sijaan vaihteli vain vähän. Tuhkalannoitus, jonka tarkoituksena on edistää puiden kasvua ojitetuilla soilla, ei merkittävästi vaikuttanut metaanintuottoon tai -tuottajiin. Ojitetun suon yhteisöt kuitenkin muuttuivat turvesyvyyden mukaan. Vertailtaessa erilaisia PCR-menetelmiä todettiin, että kolmella mcrA-geeniin kohdistuvalla alukeparilla havaittiin pääosin samat ojitetun suon metanogeenit, mutta lajien runsaussuhteet riippuvat käytetyistä alukkeista. Soilla havaitut bakteerit kuuluivat pääjaksoihin Deltaproteobacteria, Acidobacteria ja Verrucomicrobia. Lisäksi löydettiin Crenarchaeota-pääjakson ryhmiin 1.1c ja 1.3 kuuluvia ei-metanogeenisia arkkeja. Tulokset ryhmien esiintymisestä hapettomassa turpeessa antavat lähtökohdan selvittää niiden mahdollisia vuorovaikutuksia metanogeenien kanssa. Tutkimuksen tulokset osoittivat, että metanogeeniyhteisön koostumus heijastaa metaanintuottoon vaikuttavia kemiallisia tai kasvillisuuden vaihteluita kuten suotyyppiä. Soiden metanogeenien ja niiden fysiologian parempi tuntemus voi auttaa ennustamaan ympäristömuutosten vaikutusta soiden metaanipäästöihin.

Quorum sensing in the plant pathogen Erwinia carotovora subsp. carotovora

Erwinia carotovora subsp. carotovora (Ecc) is a Gram-negative enterobacterium that causes soft-rot in potato and other crops. The main virulence determinants, the extracellular plant cell wall -degrading enzymes (PCWDEs), lead to plant tissue maceration. In order to establish a successful infection the production of PCWDEs are controlled by a complex regulatory network, including both specific and global activators and repressors. One of the most important virulence regulation systems in Ecc is mediated by quorum sensing (QS), which is a population density -dependent cell-to-cell communication mechanism used by many Gram-negative bacteria. In these bacteria N-acylhomoserine lactones (AHSL), act as diffusible signaling molecules enabling communication between bacterial cells. The AHSLs are structurally diverse and differ in their acyl chain length. This gives the bacteria signaling specificity and enables the recognition and communication within its own species. In order to detect and respond to the AHSLs the bacteria use QS regulators, LuxR-type proteins. The aim of this study was to get a deeper understanding of the Ecc QS system. In the first part of the study we showed that even different strains of Ecc use different dialects and of physiological concentrations, only the cognate AHSL with the correct acyl chain is recognized as a signal that can switch on virulence genes. The molecular basis of the substrate specificity of the AHSL synthase ExpI was investigated in order to recognize the acyl chain length specificity determinants of distinct AHSL synthases. Several critical residues that define the size of the substrate-binding pocket were identified. We demonstrated that in the ExpISCC1 mutations M127T and F69L are sufficient to change the N-3-oxohexanoyl-L-homoserine lactone producing ExpISCC1 to an N-3-oxooctanoyl-L-homoserine lactone (3-oxo-C8-HSL) producing enzyme. In the second study the means of sensing specificity and response to the AHSL signaling molecule were investigated. We demonstrated that the AHSL receptor ExpR1 of Ecc strain SCC3193 has strict specificity for the cognate AHSL 3-oxo-C8-HSL. In addition we identified a second AHSL receptor ExpR2 with a novel property to sense AHSLs with different acyl chain lengths. In the absence of AHSLs ExpR1 and ExpR2 were found to act synergistically to repress the virulence gene expression. This repression was shown to be released by addition of AHSLs and appears to be largely mediated by the global negative regulator RsmA. In the third study random transposon mutagenesis was used to widen the knowledge of the Ecc QS regulon. Two new QS-controlled target genes, encoding a DNA-binding regulator Hor and a plant ferredoxin-like protein FerE, were identified. The QS control of the identified genes was executed by the QS regulators ExpR1 and ExpR2 and as expression of PCWDE genes mediated by the RsmA repressor. Hor was shown to contribute to bacterial virulence at least partly through its control of PCWDE production, while FerE was shown to contribute to oxidative stress tolerance and in planta fitness of the bacteria. In addition our results suggest that QS is central to the control of oxidative stress tolerance in Ecc. In conclusion, these results indicate that Ecc strain SCC3193 is able to react and respond both to the cognate AHSL signal and the signals produced by other bacterial species, in order to control a wide variety of functions in the plant pathogen Ecc.

Archaea in the Mycorrhizosphere of Boreal Forest Trees

Archaea were long thought to be a group of ancient bacteria, which mainly lived in extreme environments. Due to the development of DNA sequencing methods and molecular phylogenetic analyses, it was shown that the living organisms are in fact divided into three domains; the Archaea, Bacteria and the Eucarya. Since the beginning of the previous decade, it was shown that archaea generally inhabit moderate environments and that these non-extremophilic archaea are more ubiquitous than the extremophiles. Group 1 of non-extreme archaea affiliate with the phylum Crenarchaeota. The most commonly found soil archaea belong to the subgroup 1.1b. However, the Crenarchaeota found in the Fennoscandian boreal forest soil belong to the subgroup 1.1c. The organic top layer of the boreal forest soil, the humus, is dominated by ectomycorrhizal fungal hyphae. These colonise virtually all tree fine root tips in the humus layer and have been shown to harbour distinct bacterial populations different from those in the humus. The archaea have also been shown to colonise both boreal forest humus and the rhizospheres of plants. In this work, studies on the archaeal communities in the ectomycorrhizospheres of boreal forest trees were conducted in microcosms. Archaea belonging to the group 1.1c Crenarchaeota and Euryarchaeota of the genera Halobacterium and Methanolobus were detected. The archaea generally colonised fungal habitats, such as ectomycorrhizas and external mycelia, rather than the non-mycorrhizal fine roots of trees. The species of ectomycorrhizal fungus had a great impact on the archaeal community composition. A stable euryarchaeotal community was detected especially in the mycorrhizas, of most of the tested Scots pine colonising ectomycorrhizal fungi. The Crenarchaeota appeared more sporadically in these habitats, but had a greater diversity than the Euryarchaeota. P. involutus mycorrhizas had a higher diversity of 1.1c Crenarchaeota than the other ectomycorrhizal fungi. The detection level of archaea in the roots of boreal trees was generally low although archaea have been shown to associate with roots of different plants. However, alder showed a high diversity of 1.1c Crenarchaeota, exceeding that of any of the tested mycorrhizas. The archaeal 16S rRNA genes detected from the non-mycorrhizal roots were different from those of the P. involutus mycorrhizas. In the phylogenetic analyses, the archaeal 16S rRNA gene sequences obtained from non-mycorrhizal fine roots fell in a separate cluster within the group 1.1c Crenarchaeota than those from the mycorrhizas. When the roots of the differrent tree species were colonised by P. involutus, the diversity and frequency of the archaeal populations of the different tree species were more similar to each other. Both Cren- and Euryarchaeota were enriched in cultures to which C-1 substrates were added. The 1.1c Crenarchaeota grew anaerobically in mineral medium with CH4 and CO2 as the only available C sources, and in yeast extract media with CO2 and CH4 or H2. The crenarchaeotal diversity was higher in aerobic cultures on mineral medium with CH4 or CH3OH than in the anaerobic cultures. Ecological functions of the mycorrhizal 1.1c Crenarchaeota in both anaerobic and aerobic cycling of C-1 compounds were indicated. The phylogenetic analyses did not divide the detected Crenarchaeota into anaerobic and aerobic groups. This may suggest that the mycorrhizospheric crenarchaeotal communities consist of closely related groups of anaerobic and aerobic 1.1c Crenarchaeota, or the 1.1c Crenarchaeota may be facultatively anaerobic. Halobacteria were enriched in non-saline anaerobic yeast extract medium cultures in which CH4 was either added or produced, but were not detected in the aerobic cultures. They may potentially be involved in anaerobic CH4 cycling in ectomycorrhizas. The CH4 production of the mycorrhizal samples was over 10 times higher than for humus devoid of mycorrhizal hyphae, indicating a high CH4 production potential of the mycorrhizal metanogenic community. Autofluorescent methanogenic archaea were detected by microscopy and 16S rRNA gene sequences of the genus Methanolobus were obtained. The archaeal community depended on both tree species and the type of ectomycorrhizal fungus colonising the roots and the Cren- and Euryarchaeota may have different ecological functions in the different parts of the boreal forest tree rhizosphere and mycorrhizosphere. By employing the results of this study, it may be possible to isolate both 1.1c Crenarchaeota as well as non-halophilic halobacteria and aerotolerant methanogens from mycorrhizospheres. These archaea may be used as indicators for change in the boreal forest soil ecosystem due to different factors, such as exploitations of forests and the rise in global temperature. More information about the microbial populations with apparently low cell numbers but significant ecological impacts, such as the boreal forest soil methanogens, may be of crucial importance to counteract human impacts on such globally important ecosystems as the boreal forests.

Mu in vitro Transposition Technology in Functional Genetics and Genomics: Applications on Mouse and Bacteriophages

Transposons are mobile elements of genetic material that are able to move in the genomes of their host organisms using a special form of recombination called transposition. Bacteriophage Mu was the first transposon for which a cell-free in vitro transposition reaction was developed. Subsequently, the reaction has been refined and the minimal Mu in vitro reaction is useful in the generation of comprehensive libraries of mutant DNA molecules that can be used in a variety of applications. To date, the functional genetics applications of Mu in vitro technology have been subjected to either plasmids or genomic regions and entire genomes of viruses cloned on specific vectors. This study expands the use of Mu in vitro transposition in functional genetics and genomics by describing novel methods applicable to the targeted transgenesis of mouse and the whole-genome analysis of bacteriophages. The methods described here are rapid, efficient, and easily applicable to a wide variety of organisms, demonstrating the potential of the Mu transposition technology in the functional analysis of genes and genomes. First, an easy-to-use, rapid strategy to generate construct for the targeted mutagenesis of mouse genes was developed. To test the strategy, a gene encoding a neuronal K+/Cl- cotransporter was mutagenised. After a highly efficient transpositional mutagenesis, the gene fragments mutagenised were cloned into a vector backbone and transferred into bacterial cells. These constructs were screened with PCR using an effective 3D matrix system. In addition to traditional knock-out constructs, the method developed yields hypomorphic alleles that lead into reduced expression of the target gene in transgenic mice and have since been used in a follow-up study. Moreover, a scheme is devised to rapidly produce conditional alleles from the constructs produced. Next, an efficient strategy for the whole-genome analysis of bacteriophages was developed based on the transpositional mutagenesis of uncloned, infective virus genomes and their subsequent transfer into susceptible host cells. Mutant viruses able to produce viable progeny were collected and their transposon integration sites determined to map genomic regions nonessential to the viral life cycle. This method, applied here to three very different bacteriophages, PRD1, ΦYeO3 12, and PM2, does not require the target genome to be cloned and is directly applicable to all DNA and RNA viruses that have infective genomes. The method developed yielded valuable novel information on the three bacteriophages studied and whole-genome data can be complemented with concomitant studies on individual genes. Moreover, end-modified transposons constructed for this study can be used to manipulate genomes devoid of suitable restriction sites.

Stress responses of Gram-positive bacteria to cationic antimicrobial peptides

As the resistance of bacteria to conventional antibiotics has become an increasing problem, new antimicrobial drugs are urgently needed. One possible source of new antibacterial agents is a group of cationic antimicrobial peptides (CAMPs) produced by practically all living organisms. These peptides are typically small, amphipathic and positively charged and contain well defined a-helical or b-sheet secondary structures. The main antibacterial action mechanism of CAMPs is considered to be disruption of the cell membrane, but other targets of CAMPs also exist. Some bacterial species have evolved defence mechanisms against the harmful effects of CAMPs. One of the most effective defence mechanisms is reduction of the net negative charge of bacterial cell surfaces. Global analysis of gene expression of two Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus, was used to further study the stress responses induced by different types of CAMPs. B. subtilis cells were treated with sublethal concentrations of a-helical peptide LL-37, b-sheet peptide protegrin 1 or synthetic analogue poly-L-lysine, and the changes in gene expression were studied using DNA macroarrays. In the case of S. aureus, three different a-helical peptides were selected for the transcriptome analyses: temporin L, ovispirin-1 and dermaseptin K4-S4(1-16). Transcriptional changes caused by peptide stress were examined using oligo DNA microarrays. The transcriptome analysis revealed two main cell signalling mechanisms mediating CAMP stress responses in Gram-positive bacteria: extracytoplasmic function (ECF)sigma factors and two-component systems (TCSs). In B. subtilis, ECF sigma factors sigW and sigM as well as TCS LiaRS responded to the cell membrane disruption caused by CAMPs. In S. aureus, CAMPs caused a similar stress response to antibiotics interfering in cell wall synthesis, and TCS VraSR was strongly activated. All of these transcriptional regulators are known to respond to several compounds other than CAMPs interfering with cell envelope integrity, suggesting that they sense cell envelope stress in general. Among the most strongly induced genes were yxdLM (in B. subtilis) and vraDE (in S. aureus) encoding homologous ABC transporters. Transcription of yxdLM and vraDE operons is controlled by TCSs YxdJK and ApsRS, respectively. These TCSs seemed to be responsible for the direct recognition of CAMPs. The yxdLM operon was specifically induced by LL-37, but its role in CAMP resistance remained unclear. VraDE was proven to be a bacitracin transporter. We also showed that the net positive charge of the cell wall affects the signalrecognition of different TCSs responding to cell envelope stress. Inactivation of the Dlt system responsible for the D-alanylation of teichoic acids had a strong and differential effect on the activity of the studied TCSs, depending on their functional role in cells and the stimuli they sense.

Thymidine inhibits the incorporation of 5-fluoro-2'-deoxyuridine to DNA of mouse mammary tumour

5-Fluoro-2'-deoxyuricine is incorporated into DNA of mouse breast tumour Image . The incorporation is inhibited by thymidine. Part of the fluorodeoxyuridine is cleaved to fluorouracil and is incorporated into RNA. This incorporation is enhanced by thymidine. The result suggests that the major mechanism of action of the fluorouracil is due to its incorporation into RNA. FUra, 5-Fluorouracil; FdUR, 5-Fluoro-2'-deoxyuridine; FdUMP, 5-Fluoro-2'-deoxyuridine-5'-monophosphate.

Interaction of surfactants with DNA. Role of hydrophobicity and surface charge on intercalation and DNA melting

A probe, 9-(anthrylmethyl)trimethylammonium chloride, 1, was prepared. 1 binds to calf-thymus DNA or Escherichia coli genomic DNA with high affinity, as evidenced from the absorption titration. Strong hypochromism, spectral broadening and red-shifts in the absorption spectra were observed. Half-reciprocal plot constructed from this experiment gave binding constant of 5±0.5×104 M−1 in base molarity. We employed this anthryl probe-DNA complex for studying the effects of addition of various surfactant to DNA. Surfactants of different charge types and chain lengths were used in this study and the effects of surfactant addition to such probe-DNA complex were compared with that of small organic cations or salts. Addition of either salts or cationic surfactants led to structural changes in DNA and under these conditions, the probe from the DNA-bound complex appeared to get released. However, the cationic surfactants could induce such release of the probe from the probe-DNA complex at a much lower concentration than that of the small organic cations or salts. In contrast the anionic surfactants failed to promote any destabilization of such probe-DNA complexes. The effects of additives on the probe-DNA complexes were also examined by using a different technique (fluorescence spectroscopy) using a different probe ethidium bromide. The association complexes formed between the cationic surfactants and the plasmid DNA pTZ19R, were further examined under agarose gel electrophoresis and could not be visualized by ethidium bromide staining presumably due to cationic surfactant-induced condensation of DNA. Most of the DNA from such association complexes can be recovered by extraction of surfactants with phenol-chloroform. Inclusion of surfactants and other additives into the DNA generally enhanced the DNA melting temperatures by a few °C and at high [surfactant], the corresponding melting profiles got broadened.

Chemistry and biology of DNA methyltransferases

Recognition of a specific DNA sequence by a protein is probably the best example of macromolecular interactions leading to various events. It is a prerequisite to understanding the basis of protein-DNA interactions to obtain a better insight into fundamental processes such as transcription, replication, repair, and recombination. DNA methyltransferases with varying sequence specificities provide an excellent model system for understanding the molecular mechanism of specific DNA recognition. Sequence comparison of cloned genes, along with mutational analyses and recent crystallographic studies, have clearly defined the functions of various conserved motifs. These enzymes access their target base in an elegant manner by flipping it out of the DNA double helix. The drastic protein-induced DNA distortion, first reported for HhaI DNA methyltransferase, appears to be a common mechanism employed by various proteins that need to act on bases. A remarkable feature of the catalytic mechanism of DNA (cytosine-5) methyltransferases is the ability of these enzymes to induce deamination of the target cytosine in the absence of S-adenosyl-L-methionine or its analogs. The enzyme-catalyzed deamination reaction is postulated to be the major cause of mutational hotspots at CpG islands responsible for various human genetic disorders. Methylation of adenine residues in Escherichia coli is known to regulate various processes such as transcription, replication, repair, recombination, transposition, and phage packaging.

Identification and functional characterization of a cis-acting positive DNA element regulating CYP 2B1/B2 gene transcription in rat liver

A positive cis-acting DNA element in the near 5'-upstream region of the CYP2B1/B2 genes in rat liver was found to play an important role in the transcription of these genes. An oligonucleotide covering -69 to -98 nt mimicked the gel mobility shift pattern given by the fragment -179 to +29 nt, which was earlier found adequate to confer the regulatory features of this gene. Two major complexes were seen, of which the slower and faster moving complexes became intense under uninduced and Phenobarbitone-induced conditions respectively. Minigene cloned DNA plasmid covering -179 to +181 nt in pUC 19 and Bal 31 mutants derived from this parent were transcribed in whole nuclei and cell free transcription extracts and mutants containing only upto -75 nt of the upstream were poorly transcribed. Transcription extracts from phenobarbitone-injected rat liver nuclei were significantly more active than extracts from uninduced rats in transcribing the minigene constructs. Addition of the oligonucleotide (-69 to -98nt) specifically inhibited the transcription of the minigene construct (-179 to +181 nt) in the cell free transcription system. It is therefore, concluded that the region -69 to -98 nt acts as a positive cis-acting element in the transcription of the CYP2B1/B2 genes and in mediating the inductive effects of phenobarbitone.

Role of the Central Metal Ion and Ligand Charge in the DNA Binding and Modification by Metallosalen Complexes

Several metal complexes of three different functionalized salen derivatives have been synthesized. The salens differ in terms of the electrostatic character and the location of the charges. The interactions of such complexes with DNA were first investigated in detail by UV−vis absorption titrimetry. It appears that the DNA binding by most of these compounds is primarily due to a combination of electrostatic and other modes of interactions. The melting temperatures of DNA in the presence of various metal complexes were higher than that of the pure DNA. The presence of additional charge on the central metal ion core in the complex, however, alters the nature of binding. Bis-cationic salen complexes containing central Ni(II) or Mn(III) were found to induce DNA strand scission, especially in the presence of co-oxidant as revealed by plasmid DNA cleavage assay and also on the basis of the autoradiogram obtained from their respective high-resolution sequencing gels. Modest base selectivity was observed in the DNA cleavage reactions. Comparisons of the linearized and supercoiled forms of DNA in the metal complex-mediated cleavage reactions reveal that the supercoiled forms are more susceptible to DNA scission. Under suitable conditions, the DNA cleavage reactions can be induced either by preformed metal complexes or by in situ complexation of the ligand in the presence of the appropriate metal ion. Also revealed was the fact that the analogous complexes containing Cu(II) or Cr(III) did not effect any DNA strand scission under comparable conditions. Salens with pendant negative charges on either side of the precursor salicylaldehyde or ethylenediamine fragments did not bind with DNA. Similarly, metallosalen complexes with net anionic character also failed to induce any DNA modification activities.