Nonrandom location of IS1 elements in the genomes of natural isolates of Escherichia coli

We have studied the spatial distribution of IS1 elements in the genomes of natural isolates comprising the ECOR reference collection of Escherichia coli. We find evidence for nonrandomness at three levels. Many pairs of IS1 elements are in much closer proximity (< 10 kb) than can be accounted for by chance. IS1 elements in close proximity were identified by long-range PCR amplification of the genomic sequence between them. Each amplified region was sequenced and its map location determined by database screening of DNA hybridization. Among the ECOR strains with at least two IS1 elements, 54% had one or more pairs of elements separated by < 10 kb. We propose that this type of clustering is a result of "local hopping," in which we assume that a significant proportion of tranposition events leads to the insertion of a daughter IS element in the vicinity of the parental element. A second level of nonrandomness is found in strains with a modest number of IS1 elements that are mapped through the use of inverse PCR to amplify flanking genomic sequences: in these strains, the insertion sites tend to be clustered over a smaller region of chromosome than would be expected by chance. A third level of nonrandomness is observed in the composite distribution of IS elements across strains: among 20 mapped IS1 elements, none were found in the region of 48-77 minutes, a significant gap. One region of the E. coli chromosome, at 98 min, had a cluster of IS1 elements in seven ECOR strains of diverse phylogenetic origin. We deduce from sequence analysis that this pattern of distribution is a result of initial insertion in the most recent common ancestor of these strains and therefore not a hot spot of insertion. Analysis using long- range PCR with primers for IS2 and IS3 also yielded pairs of elements in close proximity, suggesting that these elements may also occasionally transpose by local hopping.      

Metabolism of lithocholic acid in the rat: formation of lithocholic acid 3-O-glucuronide in vivo

Milligram amounts of [3 beta-3H]lithocholic (3 alpha-hydroxy-5 beta-cholanoic) acid were administered by intravenous infusion to rats prepared with a biliary fistula. Analysis of sequential bile samples by thin-layer chromatography (TLC) demonstrated that lithocholic acid glucuronide was present in bile throughout the course of the experiments and that its secretion rate paralleled that of total isotope secretion. Initial confirmation of the identity of this metabolite was obtained by the recovery of labeled lithocholic acid after beta-glucuronidase hydrolysis of bile samples. For detailed analysis of biliary metabolites of [3H]lithocholic acid, pooled bile samples from infused rats were subjected to reversed-phase chromatography and four major labeled peaks were isolated. After complete deconjugation, the two major compounds in the combined first two peaks were identified as murideoxycholic (3 alpha, 6 beta-dihydroxy-5 beta-cholanoic) and beta-muricholic (3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanoic) acids and the third peak was identified as taurolithocholic acid. The major component of the fourth peak, after isolation, derivatization (to the methyl ester acetate), and purification by high pressure liquid chromatography (HPLC), was positively identified by proton nuclear magnetic resonance as lithocholic acid 3 alpha-O-(beta-D-glucuronide). These studies have shown, for the first time, that lithocholic acid glucuronide is a product of in vivo hepatic metabolism of lithocholic acid in the rat.     

Cytologic features of well-differentiated sclerosing liposarcoma in aspirated samples

The histologic, cytologic and ultrastructural features of two cases of a rare variant of liposarcoma, the well-differentiated sclerosing type, are presented. These tumors are characterized by lipoblasts (in different stages of development), atypical fibroblasts, multinucleated floret cells, lipocytes and delicate, dispersed collagen. The differential diagnosis includes other types of liposarcoma and three benign lipomatous lesions: the pleomorphic lipoma, the atypical lipoma and the spindle cell lipoma. The latter three neoplasms are composed of fibroblasts, lipocytes and coarse collagen and may have typical lipoblastlike or fibroblastic cells. Given the difficulty in distinguishing these lesions, both cytologically and histologically, the clinical presentation and the tumor location become essential in rendering the diagnosis of sclerosing liposarcoma.     

Fitness effects of amino acid replacements in the beta-galactosidase of Escherichia coli

Two genetic procedures were used to obtain amino acid replacements in the lacZ-encoded beta-galactosidase in Escherichia coli. Amino acid replacements could be obtained without regard to their effects on lactase activity by selecting spontaneous mutations that relieved the strong polarity of six nonsense mutations. When streaked on MacConkey- lactose indicator plates, approximately 75% of these mutants gave strong red lactose-fermenting colonies, and 25% gave white nonfermenting colonies. Mutants from 11 other nonsense codons were isolated directly using MacConkey-lactose indicator plates, on which positive color indication requires only 0.5% of the wildtype lactase activity. Among the total of 17 codons, 25 variant beta-galactosidases were identified using electrophoresis and thermal denaturation studies. The fitness effects of these variant beta-galactosidases were determined using competition experiments conducted with lactose as the sole nutrient limiting the growth rate in chemostat cultures. Three of the replacements were deleterious, one was selectively advantageous, and the selective effects of the remaining 21 were undetectable under conditions in which the smallest detectable selection coefficient was approximately 0.4%/generation.      

Patterns of Admixture and Population Structure in Native Populations of Northwest North America

The initial contact of European populations with indigenous populations of the Americas produced diverse admixture processes across North, Central, and South America. Recent studies have examined the genetic structure of indigenous populations of Latin America and the Caribbean and their admixed descendants, reporting on the genomic impact of the history of admixture with colonizing populations of European and African ancestry. However, relatively little genomic research has been conducted on admixture in indigenous North American populations. In this study, we analyze genomic data at 475,109 single-nucleotide polymorphisms sampled in indigenous peoples of the Pacific Northwest in British Columbia and Southeast Alaska, populations with a well-documented history of contact with European and Asian traders, fishermen, and contract laborers. We find that the indigenous populations of the Pacific Northwest have higher gene diversity than Latin American indigenous populations. Among the Pacific Northwest populations, interior groups provide more evidence for East Asian admixture, whereas coastal groups have higher levels of European admixture. In contrast with many Latin American indigenous populations, the variance of admixture is high in each of the Pacific Northwest indigenous populations, as expected for recent and ongoing admixture processes. The results reveal some similarities but notable differences between admixture patterns in the Pacific Northwest and those in Latin America, contributing to a more detailed understanding of the genomic consequences of European colonization events throughout the Americas.     

The Human-Bacterial Pathogen Protein Interaction Networks of Bacillus anthracis,Francisella tularensis,and Yersinia pestis

Background

Bacillus anthracis, Francisella tularensis, and Yersinia pestis are bacterial pathogens that can cause anthrax, lethal acute pneumonic disease, and bubonic plague, respectively, and are listed as NIAID Category A priority pathogens for possible use as biological weapons. However, the interactions between human proteins and proteins in these bacteria remain poorly characterized leading to an incomplete understanding of their pathogenesis and mechanisms of immune evasion.

Methodology

In this study, we used a high-throughput yeast two-hybrid assay to identify physical interactions between human proteins and proteins from each of these three pathogens. From more than 250,000 screens performed, we identified 3,073 human-B. anthracis, 1,383 human-F. tularensis, and 4,059 human-Y. pestis protein-protein interactions including interactions involving 304 B. anthracis, 52 F. tularensis, and 330 Y. pestis proteins that are uncharacterized. Computational analysis revealed that pathogen proteins preferentially interact with human proteins that are hubs and bottlenecks in the human PPI network. In addition, we computed modules of human-pathogen PPIs that are conserved amongst the three networks. Functionally, such conserved modules reveal commonalities between how the different pathogens interact with crucial host pathways involved in inflammation and immunity.

Significance

These data constitute the first extensive protein interaction networks constructed for bacterial pathogens and their human hosts. This study provides novel insights into host-pathogen interactions.