p53 and DNA damage checkpoint responses in the human prostate

Prostate cancer is the most common noncutaneous malignancy and the second leading cause of cancer mortality in men. In 2004, 5237 new cases were diagnosed and altogether 25 664 men suffered from prostate cancer in Finland (Suomen Syöpärekisteri). Although extensively investigated, we still have a very rudimentary understanding of the molecular mechanisms leading to the frequent transformation of the prostate epithelium. Prostate cancer is characterized by several unique features including the multifocal origin of tumors and extreme resistance to chemotherapy, and new treatment options are therefore urgently needed. The integrity of genomic DNA is constantly challenged by genotoxic insults. Cellular responses to DNA damage involve elegant checkpoint cascades enforcing cell cycle arrest, thus facilitating damage repair, apoptosis or cellular senescence. Cellular DNA damage triggers the activation of tumor suppressor protein p53 and Wee1 kinase which act as executors of the cellular checkpoint responses. These are essential for genomic integrity, and are activated in early stages of tumorigenesis in order to function as barriers against tumor formation. Our work establishes that the primary human prostatic epithelial cells and prostatic epithelium have unexpectedly indulgent checkpoint surveillance. This is evidenced by the absence of inhibitory Tyr15 phosphorylation on Cdk2, lack of p53 response, radioresistant DNA synthesis, lack of G1/S and G2/M phase arrest, and presence of persistent gammaH2AX damage foci. We ascribe the absence of inhibitory Tyr15 phosphorylation to low levels of Wee1A, a tyrosine kinase and negative regulator of cell cycle progression. Ectopic Wee1A kinase restored Cdk2-Tyr15 phosphorylation and efficiently rescued the ionizing radiation-induced checkpoints in the human prostatic epithelial cells. As variability in the DNA damage responses has been shown to underlie susceptibility to cancer, our results imply that a suboptimal checkpoint arrest may greatly increase the accumulation of genetic lesions in the prostate epithelia. We also show that small molecules can restore p53 function in prostatic epithelial cells and may serve as a paradigm for the development of future therapeutic agents for the treatment of prostate cancer We hypothesize that the prostate has evolved to activate the damage surveillance pathways and molecules involved in these pathways only to certain stresses in extreme circumstances. In doing so, this organ inadvertently made itself vulnerable to genotoxic stress, which may have implications in malignant transformation. Recognition of the limited activity of p53 and Wee1 in the prostate could drive mechanism-based discovery of preventative and therapeutic agents.

A detailed study of Li-DNA fibres at various salt concentrations reveals a non-helical B-DNA and a possible similarity of solution and solid state structures

A thorough investigation of salt concentration dependence of lithium DNA fibres is made using X-ray diffraction. While for low salt the C-form pattern is obtained, crystalline B-type diffraction patterns result on increasing the salt concentration. The salt content in the gel (from which fibres are drawn) is estimated by equilibrium dialysis using the Donnan equilibrium principle. The salt range giving the best crystalline B pattern is determined. It is found that in this range meridional reflections occur on the fourth and sixth layer lines. In addition, the tenth layer meridian is absent at a particular salt concentration. These results strongly suggest the presence of non-helical features in the DNA molecule. Preliminary analysis of the diffraction patterns indicates a structural variability within the B-form itself. Further, the possibility of the structural parameters of DNA being similar in solid state and in solution is discussed.

Sex Determination - A Hypothesis Based On Noncoding Dna

Certain recent models of sex determination in mammals, Drosophila melanogaster, Caenorhabditis elegans, and snakes are examined in the light of the hypothesis that the relevant genetic regulatory mechanisms are similar and interrelated. The proposed key element in each of these instances is a noncoding DNA sequence, which serves as a high-affinity binding site for a repressor-like molecule regulating the activity of a major "sex-determining" gene. On this basis it is argued that, in several eukaryotes, (i) certain DNA sequences that are sex-determining are noncoding, in the sense that they are not the structural genes of a sex-determining protein; (ii) in some species these noncoding sequences are present in one sex and absent in the other, while in others their copy number or accessibility to regulatory molecules is significantly unequal between the two sexes; and (iii) this inequality determines whether the embryo develops into a male or a female.

N-[2-naphthyl]-glycine hydrazide, a potent inhibitor of dna-dependent rna-polymerase of mycobacterium-tuberculosis h37rv

N-[2-Naphthyl]-glycine hydrazide has been shown for the first time as a potent inhibitor of the DNA-dependent RNA polymerase (EC 2.7.7.6) of Mycobacterium tuberculosis H37Rv. At a concentration of 10 to the power -9 M, the compound shows maximum inhibition of the enzyme, the inhibition being less at higher concentrations. It is suggested that the novel type of inhibition pattern may be due to hydrophobic interactions occurring between the molecules of the compound at higher concentrations. The finding that there is a shift in the max of the compound could also account for this phenomenon. The effect of this compound was also tested on DNA-dependent RNA polymerases from an eukaryotic fungus, Microsporum canis. At a concentration of 10 to the power-9 M it inhibits RNA polymerase II (32 percent) but not RNA polymerases I and III.

Meta-analyses identify 13 loci associated with age at menopause and highlight DNA repair and immune pathways

To newly identify loci for age at natural menopause, we carried out a meta-analysis of 22 genome-wide association studies (GWAS) in 38,968 women of European descent, with replication in up to 14,435 women. In addition to four known loci, we identified 13 loci newly associated with age at natural menopause (at P < 5 x 10(-8)). Candidate genes located at these newly associated loci include genes implicated in DNA repair (EXO1, HELQ, UIMC1, FAM175A, FANCI, TLK1, POLG and PRIM1) and immune function (IL11, NLRP11 and PRRC2A (also known as BAT2)). Gene-set enrichment pathway analyses using the full GWAS data set identified exoDNase, NF-kappaB signaling and mitochondrial dysfunction as biological processes related to timing of menopause.

Variety quality management in strawberry, using DNA genotyping as a tool

Quality management strawberry, DNA genotyping.

Recent developments in DNA evidence

DNA evidence has made a significant contribution to criminal investigations in Australia and around the world since it was widely adopted in the 1990s (Gans & Urbas 2002). The direct matching of DNA profiles, such as comparing one obtained from a crime scene with one obtained from a suspect or database, remains a widely used technique in criminal investigations. A range of new DNA profiling techniques continues to be developed and applied in criminal investigations around the world (Smith & Urbas 2012). This paper is the third in a series by the Australian Institute of Criminology (AIC) on DNA evidence. The first, published in 1990 when the technology was in its relative infancy, outlined the scientific background for DNA evidence, considered early issues such as scientific reliability and privacy and described its application in early criminal cases (Easteal & Easteal 1990). The second, published in 2002, expanded on the scientific background and discussed a significant number of Australian cases in a 12-year period, illustrating issues that had arisen in investigations, at trial and in the use of DNA in the review of convictions and acquittals (Gans & Urbas 2002). There have been some significant developments in the science and technology behind DNA evidence in the 13 years since 2002 that have important implications for law enforcement and the legal system. These are discussed through a review of relevant legal cases and the latest empirical evidence. This paper is structured in three sections. The first examines the scientific techniques and how they have been applied in police investigations, drawing on a number of recent cases to illustrate them. The second considers empirical research evaluating DNA evidence and databases and the impact DNA has on investigative and court outcomes. The final section discusses significant cases that establish legal precedent relating to DNA evidence in criminal trials where significant issues have arisen or new techniques have been applied that have not yet been widely discussed in the literature. The paper concludes by reflecting on implications for policy and practice.

DNA markers linked to the R(2) rust resistance gene in sunflower (Helianthus annuus L.) facilitate anticipatory breeding for this disease variant.

Pre-emptive breeding for host disease resistance is an effective strategy for combating and managing devastating incursions of plant pathogens. Comprehensive, long-term studies have revealed that virulence to the R (2) sunflower (Helianthus annuus L.) rust resistance gene in the line MC29 does not exist in the Australian rust (Puccinia helianthi) population. We report in this study the identification of molecular markers linked to this gene. The three simple sequence repeat (SSR) markers ORS795, ORS882, and ORS938 were linked in coupling to the gene, while the SSR marker ORS333 was linked in repulsion. Reliable selection for homozygous-resistant individuals was efficient when the three markers, ORS795, ORS882, and ORS333, were used in combination. Phenotyping for this resistance gene is not possible in Australia without introducing a quarantinable race of the pathogen. Therefore, the availability of reliable and heritable DNA-based markers will enable the efficient deployment of this gene, permitting a more effective strategy for generating sustainable commercial cultivars containing this rust resistance gene.

Reversal of handedness in DNA: A stable link between RU and LZ helices

Two types of left-handed zig-zag (LZ) helices were obtained following stereochemical guideline. They are referred to as LZ1 and LZ2 helices. LZ1 helices have conformations similar to those found in the single crystals of d(C-G)3 and d(C-G)25,6. Z-character is more prominent in LZ2 than in LZ1 helix. The conformations of a stable link between RU and LZ helical fragments are given. The link involves inverted stacking arrangement of the bases: a characteristic feature of all RL models proposed by us

A comparison of DNA pools constructed following whole genome amplification for two-stage SNP genotyping designs

Genotyping in DNA pools reduces the cost and the time required to complete large genotyping projects. The aim of the present study was to evaluate pooling as part of a strategy for fine mapping in regions of significant linkage. Thirty-nine single nucleotide polymorphisms (SNPs) were analyzed in two genomic DNA pools of 384 individuals each and results compared with data after typing all individuals used in the pools. There were no significant differences using data from either 2 or 8 heterozygous individuals to correct frequency estimates for unequal allelic amplification. After correction, the mean difference between estimates from the genomic pool and individual allele frequencies was .033. A major limitation of the use of DNA pools is the time and effort required to carefully adjust the concentration of each individual DNA sample before mixing aliquots. Pools were also constructed by combining DNA after Multiple Displacement Amplification (MDA). The MDA pools gave similar results to pools constructed after careful DNA quantitation (mean difference from individual genotyping .040) and MDA provides a rapid method to generate pools suitable for some applications. Pools provide a rapid and cost-effective screen to eliminate SNPs that are not polymorphic in a test population and can detect minor allele frequencies as low as 1% in the pooled samples. With current levels of accuracy, pooling is best suited to an initial screen in the SNP validation process that can provide high-throughput comparisons between cases and controls to prioritize SNPs for subsequent individual genotyping.

Separation of 5-methylcytosine-rich DNA using immobilized antibody

Anti-(5-methylcytosine) antibodies were immobilized on glutaraldehyde-activated Indion 48-R, a polystyrene resin with amino groups. Immobilized antibody was very stable and could be used several times without any apparent change in the initial binding capacity of the antibody-matrix. Fractions of total DNA from various animal and plant sources were retained on this column and could be eluted quantitatively with 1.0 m NaCl. The bound fraction was further characterized for its 5-methylcytosine content by restriction enzyme digestion patterns.

Comparative analysis of Tritrichomonas foetus (Riedmuller, 1928) cat genotype, T. foetus (Riedmuller, 1928) cattle genotype and Tritrichomonas suis (Davaine, 1875) at 10 DNA loci

The parasitic protists in the genus Tritrichomonas cause significant disease in domestic cattle and cats. To assess the genetic diversity of feline and bovine isolates of Tritrichomonas foetus (Riedmuller, 1928) Wenrich and Emmerson, 1933, we used 10 different genetic regions, namely the protein coding genes of cysteine proteases 1,2 and 4-9 (CP1, 2, 4-9) involved in the pathogenesis of the disease caused by the parasite. The cytosolic malate dehydrogenase 1 (MDH1) and internal transcribed spacer region 2 of the rDNA unit (ITS2) were included as additional markers. The gene sequences were compared with those of Tritrichomonas suis (Davaine. 1875) Morgan and Hawkins, 1948 and Tritrichomonas mobilensis Culberson et al., 1986. The study revealed 100% identity for all 10 genes among all feline isolates (=T. foetus cat genotype), 100% identity among all bovine isolates (=T. foetus cattle genotype) and a genetic distinctness of 1% between the cat and cattle genotypes of T. foetus. The cattle genotype of T. foetus was 100% identical to T. suis at nine loci (CP1, 2,4-8, ITS2, MDH1). At CP9, three out of four T. suis isolates were identical to the T. foetus cattle genotype, while the T. suis isolate SUI-H3B sequence contained a single unique nucleotide substitution. Tritrichomonas mobilensis was 0.4% and 0.7% distinct from the cat and cattle genotypes of T. foetus, respectively. The genetic differences resulted in amino acid changes in the CP genes, most pronouncedly in CP2, potentially providing a platform for elucidation of genotype-specific host-pathogen interactions of T. foetus. On the basis of this data we judge T. suis and T. foetus to be subjective synonyms. For the first time, on objective nomenclatural grounds, the authority of T. suis is given to Davaine, 1875, rather than the commonly cited Gruby and Delafond, 1843. To maintain prevailing usage of T. foetus, we are suppressing the senior synomym T. suis Davaine, 1875 according to Article 23.9, because it has never been used as a valid name after 1899 and T. foetus is widely discussed as the cause of bovine trichomonosis. Thus bovine, feline and porcine isolates should all be given the name T. foetus. This promotes the stability of T. foetus for the veterinary and economically significant venereal parasite causing bovine trichomonosis. (C) 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Polymorphism and conformational flexibility of DNA: right and left handed duplexes

Left handed duplexes are shown to be in agreement with the X-ray intensity data of A-, B- and D-forms of DNA. The structures are stereochemically satisfactory because they were obtained following a stereochemical guideline derived from theory and single crystal structure data of nucleic acid components. The same stereochemical guideline also led to right handed duplexes for B- and D-forms of DNA which have stereochemically preferred conformation and hence are superior to those given by Arnott and coworkers.

Possible involvement of a calcium-stimulated ATP-hydrolyzing activity associated with mycobacteriophage I3 in the DNA injection process.

Ca2+ ions are necessary for the successful propagation of mycobacteriophage I3. An assay for the phage DNA release in the presence of an isolated cell wall preparation from the host was established, and in this system Ca2+ ions also stimulated the release of DNA. The inhibition of phage DNA injection caused by Tween 80 (polyoxyethylene sorbitan monooleate), a nonionic detergent routinely used in mycobacterial cultures, was reversed by Ca2+. The presence of a phage-associated ATP-hydrolyzing activity was demonstrated. This enzyme was stimulated by Ca2+ ions and inhibited by Tween 80. From this and the behavior of the two agents at the level of DNA injection, as well as the fact that phage I3 has a contractile tail structure, we conclude that the phage-associated ATPase is involved in the DNA injection process.