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.

Mu in vitro transposition applications in protein engineering

Transposons, mobile genetic elements that are ubiquitous in all living organisms have been used as tools in molecular biology for decades. They have the ability to move into discrete DNA locations with no apparent homology to the target site. The utility of transposons as molecular tools is based on their ability to integrate into various DNA sequences efficiently, producing extensive mutant clone libraries that can be used in various molecular biology applications. Bacteriophage Mu is one of the most useful transposons due to its well-characterized and simple in vitro transposition reaction. This study establishes the properties of the Mu in vitro transposition system as a versatile multipurpose tool in molecular biology. In addition, this study describes Mu-based applications for engineering proteins by random insertional transposon mutagenesis in order to study structure-function relationships in proteins. We initially characterized the properties of the minimal Mu in vitro transposition system. We showed that the Mu transposition system works efficiently and accurately and produces insertions into a wide spectrum of target sites in different DNA molecules. Then, we developed a pentapeptide insertion mutagenesis strategy for inserting random five amino acid cassettes into proteins. These protein variants can be used especially for screening important sites for protein-protein interactions. Also, the system may produce temperature-sensitive variants of the protein of interest. Furthermore, we developed an efficient screening system for high-resolution mapping of protein-protein interfaces with the pentapeptide insertion mutagenesis. This was accomplished by combining the mutagenesis with subsequent yeast two-hybrid screening and PCR-based genetic footprinting. This combination allows the analysis of the whole mutant library en masse, without the need for producing or isolating separate mutant clones, and the protein-protein interfaces can be determined at amino acid accuracy. The system was validated by analysing the interacting region of JFC1 with Rab8A, and we show that the interaction is mediated via the JFC1 Slp homology domain. In addition, we developed a procedure for the production of nested sets of N- and C-terminal deletion variants of proteins with the Mu system. These variants are useful in many functional studies of proteins, especially in mapping regions involved in protein-protein interactions. This methodology was validated by analysing the region in yeast Mso1 involved in an interaction with Sec1. The results of this study show that the Mu in vitro transposition system is versatile for various applicational purposes and can efficiently be adapted to random protein engineering applications for functional studies of proteins.

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.

Characterisation of the DNA-polymerase-alpha-primase complex from the silk glands of Bombyx mori

Silk gland cells ofBombyx mori undergo chromosomal endoduplication throughout larval development. The DNA content of both posterior and middle silk gland nuclei increased by 300000 times the haploid genomic content, amounting to 18 rounds of replication. The DNA doubling time is approximately 48 h and 24 h during the fourth and fifth instars of larval development. However, DNA content does not change during the interim moult. Concomitant with DNA content, DNA polymerase activity also increases as development progressed. Enzyme activity is predominantly due to DNA polymerase with no detectable level of polymerase . DNA polymerase from silk gland extracts was purified to homogeneity (using a series of columns involving ionexchange, gel-filtration and affintiy chromatography), resulting in a 4000-fold increase in specific activity. The enzyme is a heterogeneous multimer of high molecular mass, and the catalytic (polymerase) activity is resident in the 180-kDa subunit. The enzyme shows a PI of 6.2 and theKm values for the dNTP vary over 5-16 . The polymerase is tightly associated with primase activity and initiates primer synthesis in the presence of ribonucleoside triphosphates on a single-stranded DNA template. The primase activity is resident in the 45-kDa subunit. The enzyme is devoid of any detectable exonuclease activity. The abundance of DNA polymerase α in silk glands and its strong association with the nuclear matrix suggest a role in the DNA endoduplication process.

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.

Microbial diversity in the municipal composting process and development of detection methods

Composting refers to aerobic degradation of organic material and is one of the main waste treatment methods used in Finland for treating separated organic waste. The composting process allows converting organic waste to a humus-like end product which can be used to increase the organic matter in agricultural soils, in gardening, or in landscaping. Microbes play a key role as degraders during the composting-process, and the microbiology of composting has been studied for decades, but there are still open questions regarding the microbiota in industrial composting processes. It is known that with the traditional, culturing-based methods only a small fraction, below 1%, of the species in a sample is normally detected. In recent years an immense diversity of bacteria, fungi and archaea has been found to occupy many different environments. Therefore the methods of characterising microbes constantly need to be developed further. In this thesis the presence of fungi and bacteria in full-scale and pilot-scale composting processes was characterised with cloning and sequencing. Several clone libraries were constructed and altogether nearly 6000 clones were sequenced. The microbial communities detected in this study were found to differ from the compost microbes observed in previous research with cultivation based methods or with molecular methods from processes of smaller scale, although there were similarities as well. The bacterial diversity was high. Based on the non-parametric coverage estimations, the number of bacterial operational taxonomic units (OTU) in certain stages of composting was over 500. Sequences similar to Lactobacillus and Acetobacteria were frequently detected in the early stages of drum composting. In tunnel stages of composting the bacterial community comprised of Bacillus, Thermoactinomyces, Actinobacteria and Lactobacillus. The fungal diversity was found to be high and phylotypes similar to yeasts were abundantly found in the full-scale drum and tunnel processes. In addition to phylotypes similar to Candida, Pichia and Geotrichum moulds from genus Thermomyces and Penicillium were observed in tunnel stages of composting. Zygomycetes were detected in the pilot-scale composting processes and in the compost piles. In some of the samples there were a few abundant phylotypes present in the clone libraries that masked the rare ones. The rare phylotypes were of interest and a method for collecting them from clone libraries for sequencing was developed. With negative selection of the abundant phylotyps the rare ones were picked from the clone libraries. Thus 41% of the clones in the studied clone libraries were sequenced. Since microbes play a central role in composting and in many other biotechnological processes, rapid methods for characterization of microbial diversity would be of value, both scientifically and commercially. Current methods, however, lack sensitivity and specificity and are therefore under development. Microarrays have been used in microbial ecology for a decade to study the presence or absence of certain microbes of interest in a multiplex manner. The sequence database collected in this thesis was used as basis for probe design and microarray development. The enzyme assisted detection method, ligation-detection-reaction (LDR) based microarray, was adapted for species-level detection of microbes characteristic of each stage of the composting process. With the use of a specially designed control probe it was established that a species specific probe can detect target DNA representing as little as 0.04% of total DNA in a sample. The developed microarray can be used to monitor composting processes or the hygienisation of the compost end product. A large compost microbe sequence dataset was collected and analysed in this thesis. The results provide valuable information on microbial community composition during industrial scale composting processes. The microarray method was developed based on the sequence database collected in this study. The method can be utilised in following the fate of interesting microbes during composting process in an extremely sensitive and specific manner. The platform for the microarray is universal and the method can easily be adapted for studying microbes from environments other than compost.

Characterization of genomic diversity in extraintestinal pathogenic Escherichia coli (ExPEC) and development of a diagnostic DNA microarray for the differentiation of ExPEC isolates causing urinary tract infections

Extraintestinal pathogenic Escherichia coli (ExPEC) represent a diverse group of strains of E. coli, which infect extraintestinal sites, such as the urinary tract, the bloodstream, the meninges, the peritoneal cavity, and the lungs. Urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC), the major subgroup of ExPEC, are among the most prevalent microbial diseases world wide and a substantial burden for public health care systems. UTIs are responsible for serious morbidity and mortality in the elderly, in young children, and in immune-compromised and hospitalized patients. ExPEC strains are different, both from genetic and clinical perspectives, from commensal E. coli strains belonging to the normal intestinal flora and from intestinal pathogenic E. coli strains causing diarrhea. ExPEC strains are characterized by a broad range of alternate virulence factors, such as adhesins, toxins, and iron accumulation systems. Unlike diarrheagenic E. coli, whose distinctive virulence determinants evoke characteristic diarrheagenic symptoms and signs, ExPEC strains are exceedingly heterogeneous and are known to possess no specific virulence factors or a set of factors, which are obligatory for the infection of a certain extraintestinal site (e. g. the urinary tract). The ExPEC genomes are highly diverse mosaic structures in permanent flux. These strains have obtained a significant amount of DNA (predictably up to 25% of the genomes) through acquisition of foreign DNA from diverse related or non-related donor species by lateral transfer of mobile genetic elements, including pathogenicity islands (PAIs), plasmids, phages, transposons, and insertion elements. The ability of ExPEC strains to cause disease is mainly derived from this horizontally acquired gene pool; the extragenous DNA facilitates rapid adaptation of the pathogen to changing conditions and hence the extent of the spectrum of sites that can be infected. However, neither the amount of unique DNA in different ExPEC strains (or UPEC strains) nor the mechanisms lying behind the observed genomic mobility are known. Due to this extreme heterogeneity of the UPEC and ExPEC populations in general, the routine surveillance of ExPEC is exceedingly difficult. In this project, we presented a novel virulence gene algorithm (VGA) for the estimation of the extraintestinal virulence potential (VP, pathogenicity risk) of clinically relevant ExPECs and fecal E. coli isolates. The VGA was based on a DNA microarray specific for the ExPEC phenotype (ExPEC pathoarray). This array contained 77 DNA probes homologous with known (e.g. adhesion factors, iron accumulation systems, and toxins) and putative (e.g. genes predictably involved in adhesion, iron uptake, or in metabolic functions) ExPEC virulence determinants. In total, 25 of DNA probes homologous with known virulence factors and 36 of DNA probes representing putative extraintestinal virulence determinants were found at significantly higher frequency in virulent ExPEC isolates than in commensal E. coli strains. We showed that the ExPEC pathoarray and the VGA could be readily used for the differentiation of highly virulent ExPECs both from less virulent ExPEC clones and from commensal E. coli strains as well. Implementing the VGA in a group of unknown ExPECs (n=53) and fecal E. coli isolates (n=37), 83% of strains were correctly identified as extraintestinal virulent or commensal E. coli. Conversely, 15% of clinical ExPECs and 19% of fecal E. coli strains failed to raster into their respective pathogenic and non-pathogenic groups. Clinical data and virulence gene profiles of these strains warranted the estimated VPs; UPEC strains with atypically low risk-ratios were largely isolated from patients with certain medical history, including diabetes mellitus or catheterization, or from elderly patients. In addition, fecal E. coli strains with VPs characteristic for ExPEC were shown to represent the diagnostically important fraction of resident strains of the gut flora with a high potential of causing extraintestinal infections. Interestingly, a large fraction of DNA probes associated with the ExPEC phenotype corresponded to novel DNA sequences without any known function in UTIs and thus represented new genetic markers for the extraintestinal virulence. These DNA probes included unknown DNA sequences originating from the genomic subtractions of four clinical ExPEC isolates as well as from five novel cosmid sequences identified in the UPEC strains HE300 and JS299. The characterized cosmid sequences (pJS332, pJS448, pJS666, pJS700, and pJS706) revealed complex modular DNA structures with known and unknown DNA fragments arranged in a puzzle-like manner and integrated into the common E. coli genomic backbone. Furthermore, cosmid pJS332 of the UPEC strain HE300, which carried a chromosomal virulence gene cluster (iroBCDEN) encoding the salmochelin siderophore system, was shown to be part of a transmissible plasmid of Salmonella enterica. Taken together, the results of this project pointed towards the assumptions that first, (i) homologous recombination, even within coding genes, contributes to the observed mosaicism of ExPEC genomes and secondly, (ii) besides en block transfer of large DNA regions (e.g. chromosomal PAIs) also rearrangements of small DNA modules provide a means of genomic plasticity. The data presented in this project supplemented previous whole genome sequencing projects of E. coli and indicated that each E. coli genome displays a unique assemblage of individual mosaic structures, which enable these strains to successfully colonize and infect different anatomical sites.

Sequence Specific Interaction between Promoter DNA and Escherichia coli RNA Polymerase: Comparative Thermodynamic Analysis with One Immobilized Partner

Sequence specific interaction between DNA and protein molecules has been a subject of active investigation for decades now. Here, we have chosen single promoter containing bacteriophage Delta D-III T7 DNA and Escherichia coli RNA polymerase and followed their recognition at the air-water interface by using the surface plasmon resonance (SPR) technique, where the movement of one of the reacting species is restricted by way of arraying them on an immobilized support. For the Langmuir monolayer studies, we used a RNA polymerase with a histidine tag attached to one of its subunits, thus making it an xcellent substrate for Ni(II) ions, while the SPR Studies were done using biotin-labeled DNA immobilized on a streptavidin-coated chip. Detailed analysis of the thermodynamic parameters as a function of concentration and temperature revealed that the interaction of RNA polymerase with T7 DNA is largely entropy driven (83 (+/- 12) kcal mol(-1)) with a positive enthalpy of 13.6 (+/- 3.6) kcal mol(-1), The free energy of reaction determined by SPR and Langmuir-Blodgett technique was -11 (+/- 2) and -15.6 kcal mol(-1), respectively. The ability of these methods to retain the specificity of the recognition process was also established.

Relative stability of DNA as a generic criterion for promoter prediction: whole genome annotation of microbial genomes with varying nucleotide base composition

The rapid increase in genome sequence information has necessitated the annotation of their functional elements, particularly those occurring in the non-coding regions, in the genomic context. Promoter region is the key regulatory region, which enables the gene to be transcribed or repressed, but it is difficult to determine experimentally. Hence an in silico identification of promoters is crucial in order to guide experimental work and to pin point the key region that controls the transcription initiation of a gene. In this analysis, we demonstrate that while the promoter regions are in general less stable than the flanking regions, their average free energy varies depending on the GC composition of the flanking genomic sequence. We have therefore obtained a set of free energy threshold values, for genomic DNA with varying GC content and used them as generic criteria for predicting promoter regions in several microbial genomes, using an in-house developed tool `PromPredict'. On applying it to predict promoter regions corresponding to the 1144 and 612 experimentally validated TSSs in E. coli (50.8% GC) and B. subtilis (43.5% GC) sensitivity of 99% and 95% and precision values of 58% and 60%, respectively, were achieved. For the limited data set of 81 TSSs available for M. tuberculosis (65.6% GC) a sensitivity of 100% and precision of 49% was obtained.

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.