Development of a qPCR for Leifsonia xyli subsp. xyli and quantification of the effects of heat treatment of sugarcane cuttings on Lxx

The main control practice of Leifsonia xyli subsp. xyli (Lxx) in sugarcane is to heat-treat cane cuttings used as planting material in an attempt to eradicate the bacterium. A real time quantitative PCR (qPCR) protocol specific for Lxx was developed to assess the effectiveness of this practice. Primers were designed from the sequence of an Lxx-specific gene and detected as few as 10−5 ng of Lxx DNA in 100 ng of plant DNA. Two experiments were conducted to quantify Lxx titers in plants of the varieties SP80-3280 and SP70-3370 originated from cuttings treated or not by immersion in hot water at 52 °C for 30 min. In the first experiment, cuttings were collected from plant canes with low Lxx titers whereas in the second they were collected from first-ratoon canes with higher titers. Lxx was quantified in leaves by qPCR 90 days after planting and was detected in 50–90% of the plants at variable titers, indicating that the 52 °C hot water treatment for 30 min was not effective in eradicating Lxx from all plants. However, in the second experiment the bacterial population was reduced, as the median number of Lxx cells was lower compared to the non-treated control. In the case of SP70-3370, the treatment also reduced the number of Lxx-infected plants considering the pooled data of the two experiments. The results indicated that although the 52 °C hot water treatment for 30 min did not completely eliminate Lxx, it can be used to reduce the pathogen population in plants propagated from canes with high Lxx titer

Structural Genomics of Minimal Organisms: Pipeline and Results

The initial objective of the Berkeley Structural Genomics Center was to obtain a near complete three-dimensional (3D) structural information of all soluble proteins of two minimal organisms, closely related pathogens Mycoplasma genitalium and M. pneumoniae. The former has fewer than 500 genes and the latter has fewer than 700 genes. A semiautomated structural genomics pipeline was set up from target selection, cloning, expression, purification, and ultimately structural determination. At the time of this writing, structural information of more than 93percent of all soluble proteins of M. genitalium is avail able. This chapter summarizes the approaches taken by the authors' center

BAC Overlap Identification Based on Bit-Vectors

Abstract. There is no software that accurately calculates the overlap of two BACs fast enough for application to thousands of cases in turn. The problems include unacceptably low speed of dynamic programming algorithms for sequences of the considered size and failure of the faster local alignment methods to identify complete sequence overlaps. Lower sequence quality at both BAC ends and internal difference blocks, being small enough to not significantly increase relative error rates but large enough to terminate local alignments, cause output of multiple overlapping local matches which do not extend to both sequence ends. Based on Myers ’ bit-vector algorithm for fast edit distance calculation, we developed the program BACOLAP, that identifies overlapping BACs just as sensitive as global dynamic programming alignment and as fast as local heuristic alignment.