The heterotrimeric Escherichia coli RecBCD enzyme comprises two helicase motors with different polarities: RecB (3'-to-5') and RecD (5'-to-3'). This superfamily I helicase is responsible for initiating DNA double-strand-break (DSB) repair in the homologous recombination pathway. We used single-molecule tethered particle motion (TPM) experiments to visualize the RecBCD helicase translocation over long single-stranded (ss) DNA (>200 nt) with no apparent secondary structure. The bead-labeled RecBCD helicases were found to bind to the surface-immobilized blunt-end DNA, and translocate along the DNA substrates containing an ssDNA gap, resulting in a gradual decrease in the bead Brownian motion. Successful observation of RecBCD translocation over a long gap in either 3'-to-5' or 5'-to-3' ssDNA direction indicates that RecBCD helicase possesses ssDNA translocase activities in both polarities. Most RecBCD active tethers showed full translocation across the ssDNA to the dsDNA region, with about 19% of enzymes dissociated from the ss/dsDNA junction after translocating across the ssDNA region. In addition, we prepared DNA substrates containing two opposite polarities (5'-to-3' and 3'-to-5') of ssDNA regions intermitted by duplex DNA. RecBCD was able to translocate across both ssDNA regions in either ssDNA orientation orders, with less than 40% of tethers dissociating when entering into the second ssDNA region. These results suggest a model that RecBCD is able to switch between ssDNA translocases and rethread the other strand of ssDNA.

EcoRI restriction fragments of genomic DNA from Clavibacter xyli subsp. xyli (CXX) were ligated with plasmid pUC18 and cloned in Escherichia coli JM109. The cloned DNA inserts from recombinant plasmids were EcoRI‐excised and labeled with non‐radioactive digoxigenin and used as probes. Ten specific DNA probes, RSD3, 15, 30, 31, 32, 35, 37, 41, 71, and 73 were selected for disease detection and pathogen differentiation. In the specificity tests, all of the 10 CXX DNA, probes differentiated Clavibacter xyli from other bacteria specifically. Seven out of the 10 CXX probes crossreacted with C. x. subsp. cynodontis (CXC) very weakly under moderate stringency wash conditions of hybridization. In the sensitivity tests, all of the 10 DNA probes detected the homologous DNA of CXX from 0.19 to 0.75 ng. To detect various cell numbers of CXX, the DNA probes detected 104 to 105 cells effectively. In Southern hybridizations, distinctly different band patterns were shown when the probes hybridized with DNA from CXX and CXC. Among these probes, RSD3, 15, 30, 31, 35, 37, and 71, efficiently detected CXX present in the sap collected from symptomless sugarcane.

E. coli RecBCD initiates homologous repair as well as degrades foreign DNA. Recognition of chi sequence (5'-GCTGGTGG-3') switches RecBCD from a destructive, nucleolytic mode into a repair-active one that promotes RecA-mediated recombination. RecBCD includes a 3'-to-5' single-stranded DNA (ssDNA) translocase in RecB subunit, a 5'-to-3' translocase in RecD, and a secondary translocase activity associated with RecBC. To understand how chi specifically affects each translocase activity, we directly visualized individual RecBCD translocating along DNA substrates containing a ssDNA gap of different polarities, with or without chi. Disappearance of RecBCD from the ssDNA signals the loss of the ssDNA translocase activity. For substrates containing a ssDNA gap that RecBCD encounters in the 3'-to-5' polarity (3'-to-5' ssDNA), wild-type RecBCD disappears from the DNA substrates with similarly high percentage, either with chi or without. This suggests that (1) the 3'-to-5' translocase in RecB is unaffected by chi and (2) it is low in processivity. With substrates containing a ssDNA gap that RecBCD encounters in the 5'-to-3' polarity (5'-to-3' ssDNA), we found that the leaving percentage increases significantly with chi, implying inactivation of the 5'-to-3' translocase of RecD upon chi recognition. Surprisingly, the RecD defective mutant RecBCD showed only ≈50 % leaving on 5'-to-3' ssDNA, directly revealing the presence of RecBC secondary translocase and its activity is unaffected by chi. Multiple ssDNA translocases within the RecBCD complex both before and after chi ensures processive unwinding of DNA substrates required for efficient recombination events.

No abstract

In this paper, high performance and intelligent intrafield interpolation for motion compensated deinterlacing algorithms are proposed and modified. We present a correction to intelligent intra-field interpolation for motion compensated deinterlacing. The main intra-field interpolation algorithms that include cubic curve fitting interpolation, fuzzy edge interpolation and sub-sampled wide vector based edge base line averaging (ELA) are discussed in this paper. The main purpose of the design is to obtain high quality results and reduce the flickers and artifacts that are often introduced by conventional motion compensated de-interlacing. Furthermore, an improved intra-field interpolation is used to produce sharp edges in regions where MV is considered unreliable. The proposed algorithm produces sharp edges in this region and does not produce annoying effects in the high spatial frequency region.