Conformation Effects of CpG Methylation on Single-Stranded DNA Oligonucleotides: Analysis of the Opioid Peptide Dynorphin-Coding Sequences

Single-stranded DNA (ssDNA) is characterized by high conformational flexibility that allows these molecules to adopt a variety of conformations. Here we used native polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy to show that cytosine methylation at CpG sites affects the conformational flexibility of short ssDNA molecules. The CpG containing 37-nucleotide PDYN (prodynorphin) fragments were used as model molecules. The presence of secondary DNA structures was evident from differences in oligonucleotide mobilities on PAGE, from CD spectra, and from formation of A-T, G-C, and non-canonical G-T base pairs observed by NMR spectroscopy. The oligonucleotides displayed secondary structures at 4°C, and some also at 37°C. Methylation at CpG sites prompted sequence-dependent formation of novel conformations, or shifted the equilibrium between different existing ssDNA conformations. The effects of methylation on gel mobility and base pairing were comparable in strength to the effects induced by point mutations in the DNA sequences. The conformational effects of methylation may be relevant for epigenetic regulatory events in a chromatin context, including DNA-protein or DNA-DNA recognition in the course of gene transcription, and DNA replication and recombination when double-stranded DNA is unwinded to ssDNA

Simple sequence repeats in prokaryotic genomes

Simple sequence repeats (SSRs) in DNA sequences are composed of tandem iterations of short oligonucleotides and may have functional and/or structural properties that distinguish them from general DNA sequences. They are variable in length because of slip-strand mutations and may also affect local structure of the DNA molecule or the encoded proteins. Long SSRs (LSSRs) are common in eukaryotes but rare in most prokaryotes. In pathogens, SSRs can enhance antigenic variance of the pathogen population in a strategy that counteracts the host immune response. We analyze representations of SSRs in >300 prokaryotic genomes and report significant differences among different prokaryotes as well as among different types of SSRs. LSSRs composed of short oligonucleotides (1–4 bp length, designated LSSR1–4) are often found in host-adapted pathogens with reduced genomes that are not known to readily survive in a natural environment outside the host. In contrast, LSSRs composed of longer oligonucleotides (5–11 bp length, designated LSSR5–11) are found mostly in nonpathogens and opportunistic pathogens with large genomes. Comparisons among SSRs of different lengths suggest that LSSR1–4 are likely maintained by selection. This is consistent with the established role of some LSSR1–4 in enhancing antigenic variance. By contrast, abundance of LSSR5–11 in some genomes may reflect the SSRs' general tendency to expand rather than their specific role in the organisms' physiology. Differences among genomes in terms of SSR representations and their possible interpretations are discussed

Post weaning diarrhea in pigs: risk factors and non‑colistin‑based control strategies

Post-weaning diarrhea (PWD) is one of the most serious threats for the swine industry worldwide. It is commonly associated with the proliferation of enterotoxigenic Escherichia coli in the pig intestine. Colistin, a cationic antibiotic, is widely used in swine for the oral treatment of intestinal infections caused by E. coli, and particularly of PWD. However, despite the effectiveness of this antibiotic in the treatment of PWD, several studies have reported high rates of colistin resistant E. coli in swine. Furthermore, this antibiotic is considered of very high importance in humans, being used for the treatment of infections due to multidrug-resistant (MDR) Gram-negative bacteria (GNB). Moreover, the recent discovery of the mcr-1 gene encoding for colistin resistance in Enterobacteriaceae on a conjugative stable plasmid has raised great concern about the possible loss of colistin effectiveness for the treatment of MDR-GNB in humans. Consequently, it has been proposed that the use of colistin in animal production should be considered as a last resort treatment only. Thus, to overcome the economic losses, which would result from the restriction of use of colistin, especially for prophylactic purposes in PWD control, we believe that an understanding of the factors contributing to the development of this disease and the putting in place of practical alternative strategies for the control of PWD in swine is crucial. Such alternatives should improve animal gut health and reduce economic losses in pigs without promoting bacterial resistance. The present review begins with an overview of risk factors of PWD and an update of colistin use in PWD control worldwide in terms of quantities and microbiological outcomes. Subsequently, alternative strategies to the use of colistin for the control of this disease are described and discussed. Finally, a practical approach for the control of PWD in its various phases is proposed