Model-based clustering and data transformations for gene expression data.

MOTIVATION: Clustering is a useful exploratory technique for the analysis of gene expression data. Many different heuristic clustering algorithms have been proposed in this context. Clustering algorithms based on probability models offer a principled alternative to heuristic algorithms. In particular, model-based clustering assumes that the data is generated by a finite mixture of underlying probability distributions such as multivariate normal distributions. The issues of selecting a 'good' clustering method and determining the 'correct' number of clusters are reduced to model selection problems in the probability framework. Gaussian mixture models have been shown to be a powerful tool for clustering in many applications. RESULTS: We benchmarked the performance of model-based clustering on several synthetic and real gene expression data sets for which external evaluation criteria were available. The model-based approach has superior performance on our synthetic data sets, consistently selecting the correct model and the number of clusters. On real expression data, the model-based approach produced clusters of quality comparable to a leading heuristic clustering algorithm, but with the key advantage of suggesting the number of clusters and an appropriate model. We also explored the validity of the Gaussian mixture assumption on different transformations of real data. We also assessed the degree to which these real gene expression data sets fit multivariate Gaussian distributions both before and after subjecting them to commonly used data transformations. Suitably chosen transformations seem to result in reasonable fits. AVAILABILITY: MCLUST is available at http://www.stat.washington.edu/fraley/mclust. The software for the diagonal model is under development. CONTACT: kayee@cs.washington.edu. SUPPLEMENTARY INFORMATION: http://www.cs.washington.edu/homes/kayee/model.

Inorganic polyphosphate in Vibrio cholerae: genetic, biochemical, and physiologic features.

Vibrio cholerae O1, biotype El Tor, accumulates inorganic polyphosphate (poly P) principally as large clusters of granules. Poly P kinase (PPK), the enzyme that synthesizes poly P from ATP, is encoded by the ppk gene, which has been cloned from V. cholerae, overexpressed, and knocked out by insertion-deletion mutagenesis. The predicted amino acid sequence of PPK is 701 residues (81.6 kDa), with 64% identity to that of Escherichia coli, which it resembles biochemically. As in E. coli, ppk is part of an operon with ppx, the gene that encodes exopolyphosphatase (PPX). However, unlike in E. coli, PPX activity was not detected in cell extracts of wild-type V. cholerae. The ppk null mutant of V. cholerae has diminished adaptation to high concentrations of calcium in the medium as well as motility and abiotic surface attachment.

The Escherichia coli starvation gene cstC is involved in amino acid catabolism.

Escherichia coli strains mutant in the starvation gene cstC grow normally in a mineral salts medium but are impaired in utilizing amino acids as nitrogen sources. They are also compromised in starvation survival, where amino acid catabolism is important. The cstC gene encodes a 406-amino-acid protein that closely resembles the E. coli ArgD protein, which is involved in arginine biosynthesis. We postulate that CstC is a counterpart of ArgD in an amino acid catabolic pathway. The cstC upstream region contains several regulatory consensus sequences. Both sigmaS and sigma54 promoters are probably involved in cstC transcription and appear to compete with each other, presumably to match cstC expression to the cellular amino acid catabolic needs.

Novel assay reveals multiple pathways regulating stress-induced accumulations of inorganic polyphosphate in Escherichia coli.

A major impediment to understanding the biological roles of inorganic polyphosphate (polyP) has been the lack of sensitive definitive methods to extract and quantitate cellular polyP. We show that polyP recovered in extracts from cells lysed with guanidinium isothiocynate can be bound to silicate glass and quantitatively measured by a two-enzyme assay: polyP is first converted to ATP by polyP kinase, and the ATP is hydrolyzed by luciferase to generate light. This nonradioactive method can detect picomolar amounts of phosphate residues in polyP per milligram of extracted protein. A simplified procedure for preparing polyP synthesized by polyP kinase is also described. Using the new assay, we found that bacteria subjected to nutritional or osmotic stress in a rich medium or to nitrogen exhaustion had large and dynamic accumulations of polyP. By contrast, carbon exhaustion, changes in pH, temperature upshifts, and oxidative stress had no effect on polyP levels. Analysis of Escherichia coli mutants revealed that polyP accumulation depends on several regulatory genes, glnD (NtrC), rpoS, relA, and phoB.

H-NS protein represses transcription of the lux systems of Vibrio fischeri and other luminous bacteria cloned into Escherichia coli.

High expression in Escherichia coli of the lux system cistron of a luminous bacteria under its own control has been accomplished only for the Vibrio fischeri lux system at high cell density. Mutation of the hns gene in E. coli has resulted in strong expression of the V. fischeri lux system at low cell density even in an rpoS-deleted strain of E. coli that emits very low levels of luminescence. The E. coli double mutant, MC4110 hns::kan rpoS::tet carrying the lux system of V. fischeri, developed high luminescence from the very early stages of cellular growth, regardless of the presence of deletion mutations in the luxI or luxR genes. Moreover, autoinducer synthesis was restored in the double mutant with the luxR-deleted system. plac-controlled V. fischeri luxCDABE genes missing luxI and luxR were dim in E. coli rpoS mutant cells, but had wild-type levels of light in the hns-deleted strain [MC4110 hns rpoS], showing that expression was independent of lux regulators in the absence of H-NS. DNA gyrase inhibitors and DNA intercalating agents also brought about the restoration of luminescence in the rpoS-deficient strain. High expression of the lux systems of Vibrio harveyi, Photobacterium leiognathi, and Xenorhabdus luminescens in E. coli MC4110 hns rpoS cells compared with that in wild-type or rpoS mutants was also accomplished. Taken together, these data suggest that the H-NS protein inhibits transcription in E. coli of the lux systems of all or most luminous bacteria at the luxC gene as well as in the luxRI region of the V. fischeri lux operon. These DNA regions are highly enriched with homopolymeric stretches of poly d(A) and poly d(T) characterizing curved DNA, a preferable site of H-NS binding. The significance of the new findings in understanding the regulatory control of the bacterial lux system is discussed.

Electronic forms: benefits drawbacks of a World Wide Web-based approach to data entry.

It has long been realized that, compared to paper-based records, electronic record systems provide many advantages in the healthcare environment, including increased availability, improved legibility, long-term accessibility, (potentially) greater completeness, data encoding, and automated decision support and analysis. In spite of these recognized benefits, collection of patient data at the point of service generally does not occur, in large part because each such effort usually requires application-specific software and hardware, and, most significantly, provider time. Given the presence of WWW browsers now available on nearly every desktop, the support and access concerns for data entry applications can be substantially lessened. Despite these advantages, there are also downsides to the use of the WWW for data entry, including user interface issues and security. At CPMC, we are currently using web-based forms to gather patient charge data from physical and occupational therapists. Benefits of this approach have included a 98.2% user compliance rate for at least weekly data entry, and the reduction of charge posting from an average of 24.3 days to 2.3 days following the date of service. Drawbacks to WWW-based applications have included increased security exposure and persistent human tendencies to enter data in batches rather than at the time of service. A final conclusion was that, in the absence of a strong central mandate, providers must perceive a clear benefit in order to be willing to learn and use a new technology.

Use of Starvation Promoters To Limit Growth and Selectively Enrich Expression of Trichloroethylene- and Phenol-Transforming Activity in Recombinant Escherichia coli.

Volume 61, no. 9, p. 3323: the title of the article should read as shown above. [This corrects the article on p. 3323 in vol. 61.].

Use of starvation promoters to limit growth and selectively enrich expression of trichloroethylene- and phenol-transforming activity in recombinant Escherichia coli [corrected].

The expression of much useful bacterial activity is facilitated by rapid growth. This coupling can create problems in bacterial fermentations and in situ bioremediation. In the latter process, for example, it necessitates addition of large amounts of nutrients to contaminated environments, such as aquifers. This approach, termed biostimulation, can be technically difficult. Moreover, the resulting in situ bacterial biomass production can have undesirable consequences. In an attempt to minimize coupling between expression of biodegradative activity and growth, we used Escherichia coli starvation promoters to control toluene monooxygenase synthesis. This enzyme complex can degrade the environmental contaminants trichloroethylene (TCE) and phenol. Totally starving cell suspensions of such strains degraded phenol and TCE. Furthermore, rapid conversions occurred in the postexponential batch or very slow growth (dilution) rate chemostat cultures, and the nutrient demand and biomass formation for transforming a given amount of TCE or phenol were reduced by 60 to 90%. Strong starvation promoters have recently been clones and characterized in environmentally relevant bacteria like Pseudomonas species; thus, starvation promoter-driven degradative systems can now be constructed in such bacteria and tested for in situ efficacy.

The putative sigma factor KatF is regulated posttranscriptionally during carbon starvation.

Transcriptional and translational 'lacZ reporter fusions were constructed to the katF gene, which encodes a putative sigma factor centrally involved in starvation-mediated general resistance in Escherichia coli. Transcription of katF was found to increase ca. twofold after carbon starvation in minimal medium. The protein fusion containing the longest fragment of katF induced ca. eightfold under the same conditions, whereas fusions to shorter segments showed only a twofold increase in expression. The protein fusion was expressed at higher levels in a strain containing a katF::Tn10 mutation, indicating katF autoregulation. The posttranscriptional regulation of katF by starvation did not require a component of the spent minimal medium. katF was also posttranscriptionally regulated during entry into late log phase in complex medium. This induction was coincident with an increase in katE transcription, suggesting that the cellular concentration of KatF directly followed the induction of the katF protein fusion.

Continuous ambulatory peritoneal dialysis in systemic amyloidosis and end-stage renal disease.

Three patients with end-stage renal failure complicating systemic amyloidosis have been treated with continuous ambulatory peritoneal dialysis for periods of 10, 14 and 18 months respectively. In each case satisfactory control of uraemia and fluid balance has been achieved.