Michel Tessier

Parallel gene expression monitoring using oligonucleotide probe arrays of multiple transcripts with an animal model of focal ischemia.

High density oligonucleotide arrays offer tremendous potential to study gene changes occurring in disease states. The authors described the first case of using a custom designed high density oligonucleotide probe array containing 750 genes to monitor the changes in mRNA transcript levels occurring after focal ischemia for a period of 3 hours. Permanent middle cerebral artery occlusion in the rat resulted in neuronal degeneration in the dorsolateral cortex and striatum over a time course of 24 hours. Comparing the changes in hybridization levels in the frontal and parietal cortices and the striatum, between the ipsilateral and contralateral sides of the brain using the probe arrays resulted in the up-regulation of 24 genes, which showed greater than a twofold change. Very few genes were found to be downregulated after the ischemic insult. Many of the immediate early genes (IEGs) such as c-fos, NGFI-A, NGFI-C, and Krox-20 were found to be robustly up-regulated in the three different regions studied. Other genes that were up-regulated in perifocal regions included Arc, Inhibin-β-A, and the phosphatases MKP-1 and MKP-3. The hybridization signal intensity from the probe arrays enabled quantification of many genes relative to one another, and robust changes in expression were obtained with very little interanimal variability. Furthermore, the authors were able to validate the increased expression of NGFI-C and Arc using in situ hybridization. This represented the first example of using high density oligonucleotide probe arrays in studying the expression of many genes in parallel and in discrete brain regions after focal ischemia.

Isolation and characterization of the carotenoid biosynthesis genes of Flavobacterium sp. strain R1534.

The Gram-negative bacterium Flavobacterium sp. strain R1534 is a natural producer of zeaxanthin. A 14 kb genomic DNA fragment of this organism has been cloned and a 5.1 kb piece containing the carotenoid biosynthesis genes sequenced. The carotenoid biosynthesis cluster consists of five genes arranged in at least two operons. The five genes are necessary and sufficient for the synthesis of zeaxanthin. The encoded proteins have significant homology to the crtE, crtB, crtY, crtI and crtZ gene products of other carotenogenic organisms. Biochemical assignment of the individual gene products was done by HPLC analysis of the carotenoid accumulation in Escherichia coli host strains transformed with plasmids carrying deletions of the Flavobacterium sp. strain R1534 carotenoid biosynthesis cluster.

Active site residue 297 of Aspergillus niger phytase critically affects the catalytic properties.

The wild‐type phytases from the Aspergillus niger strains NRRL 3135 and T213 display a three‐fold difference in specific activity (103 versus 32 U/mg protein), despite only 12 amino acid differences that are distributed all over the sequence of the protein. Of the 12 divergent positions, three are located in or close to the substrate binding site. Site‐directed mutagenesis of these residues in A. niger T213 phytase showed that the R297Q mutation (R in T213, Q in NRRL 3135) fully accounts for the differences in catalytic properties observed. Molecular modelling revealed that R297 may directly interact with a phosphate group of phytic acid. The fact that this presumed ionic interaction – causing stronger binding of substrates and products – correlates with a lower specific activity indicates that product (myo‐inositol pentakisphosphate) release is the rate‐limiting step of the reaction.

Ongoing genome reduction in Mycobacterium ulcerans.

M. ulcerans is adapting to a more stable environment.

Structure-based chimeric enzymes as an alternative to directed enzyme evolution: phytase as a test case

Thermostability is a key feature for commercially attractive variants of the fungal enzyme phytase. In an initial set of experiments, we restored ionic interactions and hydrogen bonds on the surface of Aspergillus terreus phytase, which are present in the homologous but more thermostable enzyme from A. niger. Since these mutations turned out to be neutral, we replaced-in the same region and based on the crystal structure of A. niger phytase-entire secondary structure elements. The replacement of one alpha-helix on the surface of A. terreus phytase by the corresponding stretch of A. niger phytase resulted in an enzyme with improved thermostability and unaltered enzymatic activity. Surprisingly, the thermostability of this hybrid protein was very similar to that of A. niger phytase, although the fusion protein contained only a 31 amino acid stretch of the more stable parent enzyme. This report provides evidence that structure-based chimeric enzymes can be used to exploit the evolutionary information within a sequence alignment. We propose this method as an alternative to directed enzyme evolution if due to expression constraints the screening of large mutant populations is not feasible.

A longitudinal study of gene expression in healthy individuals

BackgroundThe use of gene expression in venous blood either as a pharmacodynamic marker in clinical trials of drugs or as a diagnostic test requires knowledge of the variability in expression over time in healthy volunteers. Here we defined a normal range of gene expression over 6 months in the blood of four cohorts of healthy men and women who were stratified by age (22–55 years and > 55 years) and gender.MethodsEleven immunomodulatory genes likely to play important roles in inflammatory conditions such as rheumatoid arthritis and infection in addition to four genes typically used as reference genes were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), as well as the full genome as represented by Affymetrix HG U133 Plus 2.0 microarrays.ResultsGene expression levels as assessed by qRT-PCR and microarray were relatively stable over time with ~2% of genes as measured by microarray showing intra-subject differences over time periods longer than one month. Fifteen genes varied by gender. The eleven genes examined by qRT-PCR remained within a limited dynamic range for all individuals. Specifically, for the seven most stably expressed genes (CXCL1, HMOX1, IL1RN, IL1B, IL6R, PTGS2, and TNF), 95% of all samples profiled fell within 1.5–2.5 Ct, the equivalent of a 4- to 6-fold dynamic range. Two subjects who experienced severe adverse events of cancer and anemia, had microarray gene expression profiles that were distinct from normal while subjects who experienced an infection had only slightly elevated levels of inflammatory markers.ConclusionThis study defines the range and variability of gene expression in healthy men and women over a six-month period. These parameters can be used to estimate the number of subjects needed to observe significant differences from normal gene expression in clinical studies. A set of genes that varied by gender was also identified as were a set of genes with elevated expression in a subject with iron deficiency anemia and another subject being treated for lung cancer.