John J. Fawcett

Pathogenomic Sequence Analysis of Bacillus cereus and Bacillus thuringiensis Isolates Closely Related to Bacillus anthracis

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are closely related gram-positive, spore-forming bacteria of the B. cereus sensu lato group. While independently derived strains of B. anthracis reveal conspicuous sequence homogeneity, environmental isolates of B. cereus and B. thuringiensis exhibit extensive genetic diversity. Here we report the sequencing and comparative analysis of the genomes of two members of the B. cereus group, B. thuringiensis 97-27 subsp. konkukian serotype H34, isolated from a necrotic human wound, and B. cereus E33L, which was isolated from a swab of a zebra carcass in Namibia. These two strains, when analyzed by amplified fragment length polymorphism within a collection of over 300 of B. cereus, B. thuringiensis, and B. anthracis isolates, appear closely related to B. anthracis. The B. cereus E33L isolate appears to be the nearest relative to B. anthracis identified thus far. Whole-genome sequencing of B. thuringiensis 97-27and B. cereus E33L was undertaken to identify shared and unique genes among these isolates in comparison to the genomes of pathogenic strains B. anthracis Ames and B. cereus G9241 and nonpathogenic strains B. cereus ATCC 10987 and B. cereus ATCC 14579. Comparison of these genomes revealed differences in terms of virulence, metabolic competence, structural components, and regulatory mechanisms.

Construction and characterization of partial digest DNA libraries made from flow-sorted human chromosome 16☆

In this report, we present the techniques used for the construction of chromosome-specific partial digest libraries from flow-sorted chromosomes and the characterization of two such libraries from human chromosome 16. These libraries were constructed to provide materials for use in the development of a high-resolution physical map of human chromosome 16, and as part of a distributive effort on the National Laboratory Gene Library Project. Libraries with 20-fold coverage were made in Charon-40 (LA16NL03) and in sCos-1 (LA16NC02) after chromosome 16 was sorted from a mouse-human monochromosomal hybrid cell line containing a single homologue of human chromosome 16. Both libraries are approximately 90% enriched for human chromosome 16, have low nonrecombinant backgrounds, and are highly representative for human chromosome-16 sequences. The cosmid library in particular has provided a valuable resource for the isolation of coding sequences, and in the ongoing development of a physical map of human chromosome 16.

Polyamine buffer for bivariate human flow cytogenetic analysis and sorting.

Publisher Summary Of the three chromosomes isolation protocols included in the methods monograph, the polyamine buffer is best suited for obtaining high molecular weight chromosomal DNA. The chromosome-stabilizing buffer uses the polyamines spermine and spermidine to stabilize chromosome structure. This program requires a massive chromosome-sorting effort that yields high molecular weight DNA for cloning. The principles of chromosome isolation using the polyamine buffer are similar to those used with the hypotonic KCl procedure and the MgSO 4 procedure. This protocol has wide applicability; however, as with other protocols, some of the variables must be optimized for each cell type––that is, blocking time, cell concentration, type of hypotonic swelling buffer, swelling time, volume of hypotonic buffer, and vortexing time. The values provided work well for normal human fibroblasts and other cell types, including somatic cell hybrids.

Chapter 19 Large-Scale Chromosome Sorting

Publisher Summary This chapter focuses on large-scale chromosome sorting for constructing large insert chromosome-specific libraries using cloning vectors such as Charon 40, cosmids, and yeast artificial chromosomes (YACs). Up to 2 μg of sorted chromosomal DNA is commonly used for constructing YAC libraries. This requires the accumulation of 4 to 20 million chromosomes. Requirements for production sorting differ from and are more demanding than the requirements of analytical sorting that typically requires only a few minutes of actual sorting time. For successful production sorting, additional precautions are needed to assure high purity, uninterrupted sorting, and recovery of high-molecular-weight DNA. Chromosome sorting is typically accomplished using chromosomes suspended in a stabilizing buffer and stained with two DNA binding fluorochromes; Hoechst 33258 and chromomycin A3. The binding specificity of each fluorochrome allows one to measure the ratio of adenine-thymine base pairs (A:T) to guanine-cytosine base pairs (G:C) and thereby resolve many more chromosomes in complex karyotypes such as human or mouse than is possible with single fluorochrome staining.