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P M Gillevet

Publications and source records attributed to P M Gillevet.

14 recordsLinked to original sources
PubMed2002

Microbial diversity and complexity in hypersaline environments: a preliminary assessment.

The microbial communities in solar salterns and a soda lake have been characterized using two techniques: BIOLOG, to estimate the metabolic potential, and amplicon length heterogeneity analysis, to estimate the molecular diversity of these communities. Both techniques demonstrated that the halophilic Bacteria and halophilic Archaea populations in the Eilat, Israel saltern are dynamic communities with extensive metabolic potentials and changing community structures. Halophilic Bacteria were detected in Mono Lake and the lower salinity ponds at the Shark Bay saltern in Western Australia, except when the crystallizer samples were stressed by exposure to Acid Green Dye #9899. At Shark Bay, halophilic Archaea were found only in the crystallizer samples. These data confirm both the metabolic diversity and the phylogenetic complexity of the microbial communities and assert the need to develop more versatile media for the cultivation of the diversity of bacteria in hypersaline environments.

Archaea↗
PubMed2000

A molecular reassessment of the Leptomyxid amoebae.

Leptomyxid amoebae encompass a diverse assemblage of amoeboid protists that have been implicated as encephalitis-causing agents. This characteristic is attributed to recent studies identifying new members of the Leptomyxidae, in particular, Balamuthia mandrillaris, that cause the disease. Their morphologies range from limax to plasmodial, as well as reticulated and polyaxial. Although systematic studies have identified B. mandrillaris as a new member of the Leptomyxidae, its precise placement within the leptomyxids is uncertain. To further assess the taxonomic placement of Balamuthia among the leptomyxid amoebae and to determine whether the members of the Leptomyxida form a monophyletic assemblage, we have sequenced 16S-like rRNA genes from representatives of three leptomyxid families. Our phylogenetic analyses revealed that current members of the order Leptomyxida do not constitute a monophyletic assemblage. Our analyses clearly show that Gephyramoeba, as well as Balamuthia do not belong in the order Leptomyxida. We highlight where molecular data give differing insights than taxonomic schemes based on traditional characters.

Amoeba↗
PubMed1998

Impact energy measurement in time-of-flight mass spectrometry with cryogenic microcalorimeters.

Time-of-flight mass spectrometry-most notably matrix-assisted laser-desorption-ionization time-of-flight (MALDI-TOF) spectrometry-is an important class of techniques for the study of proteins and other biomolecules. Although these techniques provide excellent performance for masses up to about 20,000 daltons, there has been limited success in achieving good mass resolution at higher masses. This is because the sensitivity of the microchannel plate (MCP) detectors used in most systems decreases rapidly with increasing particle mass, limiting the utility of MCP detectors for very large masses. It has recently been proposed that cryogenic particle detectors may provide a solution to these difficulties. Cryogenic detectors measure the thermal energy deposited by the particle impact, and thus have a sensitivity that is largely independent of particle mass. Recent experiments have demonstrated the sensitivity of cryogenic particle detectors to single biomolecules, a quantum efficiency several orders of magnitude larger than the MCP detectors, and sensitivity to masses as large as 750,000 daltons. Here we present results demonstrating an order of magnitude better energy resolution than previous measurements, allowing direct determination of particle charge state during acceleration. Although application of these detectors to practical mass spectrometry will require further development of the detectors and cryogenics, these detectors can be used to elucidate the performance-limiting processes that occur in such systems.

Animals↗
PubMed1998

A highly selective PCR protocol for detecting 16S rRNA genes of the genus Pseudomonas (sensu stricto) in environmental samples.

Pseudomonas species are plant, animal, and human pathogens; exhibit plant pathogen-suppressing properties useful in biological control; or express metabolic versatilities valued in biotechnology and bioremediation. Specific detection of Pseudomonas species in the environment may help us gain a more complete understanding of the ecological significance of these microorganisms. The objective of this study was to develop a PCR protocol for selective detection of Pseudomonas (sensu stricto) in environmental samples. Extensive database searches identified a highly selective PCR primer pair for amplification of Pseudomonas 16S rRNA genes. A protocol that included PCR amplification and restriction analysis, a general cloning and sequencing strategy, and phylogenetic analyses was developed. The PCR protocol was validated by testing 50 target and 14 nontarget pure cultures, which confirmed the selectivity to 100%. Further validation used amplification of target sequences from purified bulk soil DNA followed by cloning of PCR products. Restriction analysis with HaeIII revealed eight different fragmentation patterns among 36 clones. Sequencing and phylogenetic analysis of 8 representative clones indicated that 91.7% of the products were derived from target organisms of the PCR protocol. Three patterns, representing only 8.3% of the 36 clones, were derived from non-Pseudomonas or chimeric PCR artifacts. Three patterns, representing 61.1% of the clones, clustered with sequences of confirmed Pseudomonas species, whereas two patterns, representing 30.6% of the clones, formed a novel phylogenetic cluster closely associated with Pseudomonas species. The results indicated that the Pseudomonas-selective PCR primers were highly specific and may represent a powerful tool for Pseudomonas population structure analyses and taxonomic confirmations.

DNA Primers↗
PubMed1996

Sequencing homopolymer tracts and repetitive elements.

We investigated the use of Taq dye primer and Taq terminator sequencing chemistry to optimize the quality of sequence data obtained from templates containing homopolymer tracts and repetitive elements. In direct side-by-side comparisons using the Applied Biosystems Model 373A Fluorescent Sequencer, the Taq terminator sequencing chemistry gave much cleaner and more consistent results on long homopolymer tracts and dinucleotide repeats. We also investigated various thermal cycling conditions and determined that higher annealing temperatures and longer denaturation times improved the ability to sequence through these problem templates.

Base Sequence↗
PubMed1995

Exploring the Mycoplasma capricolum genome: a minimal cell reveals its physiology.

We report on the analysis of 214kb of the parasitic eubacterium Mycoplasma capricolum sequenced by genomic walking techniques. The 287 putative proteins detected to date represent about half of the estimated total number of 500 predicted for this organism. A large fraction of these (75%) can be assigned a likely function as a result of similarity searches. Several important features of the functional organization of this small genome are already apparent. Among these are (i) the expected relatively large number of enzymes involved in metabolic transport and activation, for efficient use of host cell nutrients; (ii) the presence of anabolic enzymes; (iii) the unexpected diversity of enzymes involved in DNA replication and repair; and (iv) a sizeable number of orthologues (82 so far) in Escherichia coli. This survey is beginning to provide a detailed view of how M. capricolum manages to maintain essential cellular processes with a genome much smaller than that of its bacterial relatives.

Amino Acid Sequence↗
PubMed1995

Large-scale genomic sequencing: optimization of genomic chemical sequencing reactions.

We have developed a streamlined, reproducible method for performing genomic chemical sequencing reactions on the genomic DNA of Mycoplasma capricolum, which has a genome size of about 750,000 base pairs and whose composition is 75% AT. The general modifications that ensure reproducibility and allow the processing of multiple samples can be widely adopted to other large-scale sequencing projects, while the specific modifications to the chemical reactions are applicable to the sequencing of other DNAs with a high AT content.

Adenine↗
PubMed1994

The genetic data environment an expandable GUI for multiple sequence analysis.

An X-Windows-based graphic user interface is presented which allows the seamless integration of numerous existing biomolecular programs into a single analysis environment. This environment is based on a core multiple sequence editor that is linked to external programs by a user-expandable menu system and is supported on Sun and DEC workstations. There is no limitation to the number of external functions that can be linked to the interface. The length and number of sequences that can be handled are limited only by the size of virtual memory present on the workstation. The sequence data itself is used as the reference point from which analysis is done, and scalable graphic views are supported. It is suggested that future software development utilizing this expandable, user-defined menu system and the I/O linkage of external programs will allow biologists to easily integrate expertise from disparate fields into a single environment.

Algorithms↗
PubMed1990

Purification and characterization of bovine placental retinol-binding protein.

A major low mol wt acidic protein, 3B3, produced from cultures of day 29-90 bovine allantoic membranes (in the presence of [3H]leucine or [35S]methionine) and from day 29-60 allantoic fluid, has been purified. The protein consisted of three isoelectric variants (pI 5.3-6.1) of identical mol wt (23,200 +/- 900) when analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal sequence analysis of 3B3 isolated from allantoic fluid on the first 43 amino acids showed that 3B3 had 93% and 91% homology with rabbit and human plasma retinol-binding protein (RBP), respectively. The UV absorption spectrum and the fluorescence excitation and emission spectra of purified 3B3 from both sources indicated the presence of bound retinol. Rabbit antiserum was raised against placental RBP (3B3) isolated from allantoic membrane culture medium. Placental RBP was immunoprecipitated from radiolabeled allantois and chorion culture medium and was detected in allantoic membrane culture medium and allantoic fluid by Western blotting. These results suggested that bovine placental membranes secrete RBP into allantoic fluid and that placental RBP may play important roles in vitamin A metabolism in the developing embryo.

Allantois↗
PubMed1983

Phosphorylation of proteolytically-nicked rat hepatic acetyl-CoA carboxylase.

A partially-purified preparation of acetyl-CoA carboxylase was not inactivated by ATP and Mg2+ although it was phosphorylated. SDS gel electrophoresis of the phosphorylated enzyme showed phosphopeptides migrating at 140 and 40 K along with the 250 K native subunit. Phosphorylation by the catalytic subunit of cAMP-dependent protein kinase further phosphorylated an additional 120 K phosphopeptide. Neither cAMP-independent phosphorylation nor the cAMP-dependent phosphorylation of the enzyme resulted in a significant decrease in activity.

Acetyl-CoA Carboxylase↗
PubMed1982

Rat-liver fatty-acid-synthesizing complex.

Under conditions favoring lipogenesis, a high-molecular-weight species of acetyl-CoA carboxylase was isolated that did not co-sediment with the in vitro polymerized enzyme. Assays for ATP-citrate lyase, acetyl-CoA carboxylase, and fatty acid synthetase indicated that all three enzymes were associated together as a high-molecular-weight complex and that under low-lipogenic conditions the level of these enzymes decreased. Phosphorylation of the isolated complex shifted it toward a lower molecular weight.

ATP Citrate (pro-S)-Lyase↗