Primary Base Sequence Research Articles

Single‐molecule quantification of 5‐methylcytosine and 5‐hydroxymethylcytosine in cancer genome

AbstractEpigenetic modifications present a great influence on tumorigenesis, and have attracted a growing body of researchers’ interests. They deal with genomic information to regulate biological pathways related to tumorigenesis and cancer development, without changing the primary base sequence of DNA. As the most important epigenetic modifications, 5‐methylcytosine (5mC) and 5‐hydroxmethylcytosine (5hmC) are the ones most widely investigated in human cancers. To expand our understanding of the roles of 5mC and 5hmC in cancers, multiple methods have been developed to detect and quantify 5mC and 5hmC. While various detection methods having been reviewed, here we focus on ultrasensitive single‐molecule techniques used in quantifying 5mC and 5hmC, including nanopore sequencing, single‐molecule real‐time sequencing, optical mapping, and single‐molecule fluorescent imaging.

Carcass Production and Single Nucleotide Polymorphism Adipocyte Fatty Acid Binding Protein (A-Fabp) Gene on Cairina moschata

The aim of this study was to determine differences in growth, carcass production and identify polymorphisms of adipocyte fatty acid binding protein (A-FABP) gene in Muscovy ducks from the second generation selection (G2). The research material used 180-day-old Muscovy ducks consisting of male and female ducks with white feathers and male and female ducks with a combination of black and white feathers. Measurement of duck body weight was carried out every week, and ducks are slaughtered at 10 weeks to obtain carcass production data. The data obtained were analyzed by systat-13 program based on variance analysis and Duncan test. The primary design was based on a database of the genebank Cairina moschata adipocyte fatty acid binding protein (A-FABP) gene, exons 1, 2 and partial cds (FJ763338.1). The primary base sequence of the A-FABP gene was the primary forward: 5’-TCTGGGGGTGTTATCTGGAG -3’ and reverse primer: 5’-ATTTGTCAGTGGCTGTGCTG -3’. The sequencing results of PCR products were analyzed using bioedit version 7.7 to determine the presence of the A-FABP gene polymorphism. The results showed that at the same age male Muscovy ducks produced carcass weight, and thickness of breast meat higher than female ducks. Body weight, carcass weight and parts of the carcass (breast, thigh, back, and wings) of a combination black-white feather male ducks higher than the male white feathers. The abdominal fat on all the ducks relatively the same. The A-FABP gene PCR product was at 176 bp. The results of bioedit analysis showed that at 151 bp, base length there was a mutation from Guanin to Adenin in the observed Cairina moschata, both male and female Muscovy ducks with white feathers and black-white combinations. All ducks observed had homozygous AA genotypes. Base changes in SNP c. 151G> A indicate a transition mutation. The study concluded that male Muscovy duck with a combination black-white feathers have highest genetic potential in body weight and carcass production with thick meat breast compared to other ducks. The weight of abdominal fat was relatively the same in male and female manila ducks. The A-FABP gene in manila ducks was monomorphic.

Epigenetic Therapeutics: A New Weapon in the War Against Cancer

The past 15 years have seen an explosion of discoveries related to the cellular regulation of phenotypes through epigenetic mechanisms. This regulation provides a software that packages DNA, without changing the primary base sequence, to establish heritable patterns of gene expression. In cancer, many aspects of the epigenome, controlled by DNA methylation, chromatin, and nucleosome positioning, are altered as one means by which tumor cells maintain abnormal states of self-renewal at the expense of normal maturation. Epigenetic and genetic abnormalities thus collaborate in cancer initiation and progression, as exemplified by frequent mutations in genes encoding proteins that control the epigenome. There is growing emphasis on using epigenetic therapies to reprogram neoplastic cells toward a normal state. Many agents targeting epigenetic regulation are under development and entering clinical trials. This review highlights the promise that epigenetic therapy, often in combination with other therapies, will become a potent tool for cancer management over the next decade.

Hypoxia and environmental epigenetics.

Epigenetics refers to long-term modifications of gene activity that can be inherited, either somatically or transgenerationally, but that are independent of alterations in the primary base sequence of the organism's DNA. These changes can include chemical modifications of both the DNA bases and the proteins that associate with the DNA helices to form chromatin, the nucleic acid:protein complex of which the chromosomes are comprised. Epigenetic modifications can affect the accessibility of the DNA for transcription factors (the DNA-binding proteins that specify which genes are to be active or silent by modulating the recruitment of the transcriptional machinery that reads the information encoded in the sequence) and thereby regulate the expression of genes and alter the phenotype of the organism. Epigenetic marks can also be re-established following mitosis, allowing patterns of differential gene expression to be transmitted from one cell generation to the next, and can even be maintained through meiosis, allowing transgenerational transfer of regulatory cues. Unlike the information encoded in the DNA sequence, which is invariant between most cell types and over time, epigenetic information is tissue specific and can change in response to exogenous and endogenous perturbations. This responsive capacity enables a sensitive and reactive system that can optimize gene expression in relevant tissue in response to environmental change. The realization that organisms are capable of genetically 'reprograming' themselves as well as 'preprograming' future cells, and even future offspring to optimize gene expression for a given environment may have tremendous ramifications on our understanding of both acclimatization and adaptation to hypoxia.

Solar cycle predicts folate-sensitive neonatal genotypes at discrete phases of the first trimester of pregnancy: A novel folate-related human embryo loss hypothesis

Folate, a key periconceptional nutrient, is ultraviolet light (UV-R) sensitive. We therefore hypothesise that a relationship exists between sunspot activity, a proxy for total solar irradiance (particularly UV-R) reaching Earth, and the occurrence of folate-sensitive, epigenomic-related neonatal genotypes during the first trimester of pregnancy. Limited data is provided to support the hypothesis that the solar cycle predicts folate-related human embryo loss: 379 neonates born at latitude 54°N between 1998 and 2000 were examined for three folate-sensitive, epigenome-related polymorphisms, with solar activity for trimester one accessed via the Royal Greenwich Observatory-US Air force/National Oceanic and Atmospheric Administration Sunspot Database (34,110 total observation days). Logistic regression showed solar activity predicts C677T-methylenetetrahydrofolate reductase (C677T-MTHFR) and A66G-methionine synthase reductase (A66G-MSR) genotype at discrete phases of trimester one. Total and maximal sunspot activity predicts C677T-MTHFR genotype for days 31–60 of trimester one (p=0.0181 and 0.0366, respectively) and A66G-MSR genotype for days 61–90 of trimester one (p=0.0072 and 0.0105, respectively).Loss of UV-R sensitive folate associated with the sunspot cycle might therefore interact with variant folate genes to perturb DNA methylation and/or elaboration of the primary base sequence (thymidylate synthesis), as well as increase embryo-toxic homocysteine. We hypothesise that this may influence embryo viability leading to 677CC-MTHFR and 66GG-MSR embryo loss at times of increased solar activity. This provides an interesting and plausible link between well recognised ‘folate gene originated developmental disorders’ and ‘solar activity/seasonality modulated developmental disorders’.

Information content and complexity in the high-order organization of DNA.

Nucleic acids are characterized by a vast structural variability. Secondary structural conformations include the main polymorphs A, B, and Z, cruciforms, intrinsic curvature, and multistranded motifs. DNA secondary motifs are stabilized and regulated by the primary base sequence, contextual effects, environmental factors, as well as by high-order DNA packaging modes. The high-order modes are, in turn, affected by secondary structures and by the environment. This review is concerned with the flow of structural information among the hierarchical structural levels of DNA molecules, the intricate interplay between the various factors that affect these levels, and the regulation and physiological significance of DNA high-order structures.

The 93D (hsr-omega) locus of Drosophila: non-coding gene with house-keeping functions.

The 93D, or hsr-omega (heat-shock RNA-omega), locus of Drosophila melanogaster and other species of Drosophila, besides being induced as a member of the heat shock gene family, is also selectively and singularly inducible by a variety of agents, notably benzamide, colchicine and vitamin B6 (in species other than D. melanogaster). The genomic structure of this locus is highly conserved in all species, although the primary base sequence has diverged rapidly between species. Three transcripts (two nuclear and one cytoplasmic) are produced by this locus but none of them has any significant protein coding capacity. The profile of the three transcripts varies in a developmental and inducer-specific manner. This locus is developmentally active in nearly all cell types and is essential for viability of flies. Its induction during heat shock is independent of the other members of the heat shock gene family. The other selective inducers act on this locus through separate response elements. hsr-omega activity has a characteristic effect on transcription/turnover of the heat shock induced hsp70 and the alpha-beta transcripts in D. melanogaster. It appears that the hsr-omega locus has important house-keeping functions in transport and turnover of some transcripts and in monitoring the 'health' of the translational machinery of the cell.

Methylation of cytosine influences the DNA structure.

In the past decade it has become increasingly clear that DNA has the potential to adopt a variety of unusual secondary structures which deviate from the classical right-handed B-DNA form. Intrinsic properties of the DNA molecule (primary base sequence and base modifications, degree of supercoiling) determine whether such an unusual structure is possible. Environmental factors (like ionic strength or pH of the medium, temperature, solvent polarity, interactions with proteins or drugs) have a strong influence on whether the structure is actually formed or not.

The translation of DNA primary base sequence into three-dimensional structure.

A procedure is outlined to obtain a reliable computer-generated representation of the DNA duplex from its primary sequence of base pairs. The calculations are based on the potential energies of interaction of adjacent side groups. The methods are, however, completely general and can be adapted to any set of base sequence dependent conformational rules. Static representations of the DNA are compared with the distributions of conformations obtained from Monte Carlo simulation studies. Direct matrix generator calculations of the average (equilibrium) extension and orientation of various sequences and numerical estimates of the flexibility of the chains as a whole are also reported. The methods are applied to three short fragments of kinetoplast DNA from Crithidia fasciculata which exhibit dramatically different behavior on non-denaturing polyacrylamide gels.

Four small Drosophila heat shock proteins are related to each other and to mammalian alpha-crystallin.

The primary base sequence of the protein coding regions of the four small heat shock genes of Drosophila melanogaster present at cytological locus 67B has been determined. A single open reading frame large enough to encode a small heat shock protein is found for each gene. The molecular weights of the predicted proteins are in good agreement with experimentally determined values obtained from gel electrophoresis. The predicted amino acid sequences of the four small heat shock genes show striking homologies over approximately 50% of their lengths. This region of extensive homology extends from about amino acid 85 to amino acid 195 out of a total of approximately 200 amino acids. Comparison of the predicted sequence with the known sequences of other proteins revealed a remarkable similarity between this region of homology and the corresponding region of mammalian alpha-crystallin. The possible functional significance of this structural similarity is discussed.

Comparative hybridization of mitochondrial and cytoplasmic aminoacyl transfer RNA with mitochondrial DNA from rat liver.

We have tested the specificity of hybridization of mitochondrial tRNA with mitochondrial DNA. Techniques are now available for performing RNA-DNA hybridization at low temperatures in the presence of 50 per cent formamide. With this technique, specific aminoacyl-tRNA-DNA hybridization can be followed in the presence of other species of RNA if the tRNA is acylated with radioactive amino acids. With this technique, mitochondrial leucyl-tRNA from rat liver has been found to anneal to mitochondrial DNA. Competitive hybridization between mitochondrial leucyl-tRNA and mitochondrial or cytoplasmic aminoacyl-tRNA shows that mitochondrial tRNA competes much more efficiently than cytoplasmic tRNA for sites of annealing to mitochondrial DNA. These results show that mitochondrial leucyl-tRNA differs from cytoplasmic leucyl-tRNA in its primary base sequence. They also imply that mitochondrial DNA may be the template from which mitochondrial tRNA is transcribed.