Target Site Recognition by a Diversity-Generating Retroelement

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification

Neuropathic pain in multiple sclerosis

Multiple sclerosis (MS) is a chronic neurologic disease characterized by autoimmune destruction of myelin, a sheath-like material that insulates and protects nerve fibers. A significant portion of patients with MS experience severe chronic pain that is neuropathic in nature. Preclinical studies have recently demonstrated that a specific degradation product of myelin basic protein (MBP), one of the major targets of the autoimmune response in MS, is capable of producing neuropathic pain. In particular, MBP cryptic antigenic epitope, which is hidden in intact MBP, is exposed and released postnerve injury by the activity of matrix metalloproteinase 9 (MMP-9) and produces neuropathic pain. The purpose of our study is to determine if levels of the degradation product of MBP and antibodies against it correlate with the presence and severity of chronic neuropathic pain in patients with MS as well as to find out if levels of matrix metalloproteinase 9 in urine can be used as a marker of pain in MS patients