Integrating gene expression and GO classification for PCA by preclustering

Background  

Gene expression data can be analyzed by summarizing groups of individual gene expression profiles based on GO annotation information. The mean expression profile per group can then be used to identify interesting GO categories in relation to the experimental settings. However, the expression profiles present in GO classes are often heterogeneous, i.e., there are several different expression profiles within one class. As a result, important experimental findings can be obscured because the summarizing profile does not seem to be of interest. We propose to tackle this problem by finding homogeneous subclasses within GO categories: preclustering.     

Association of polyaminergic loci with anxiety, mood disorders, and attempted suicide

Background

The polyamine system has been implicated in a number of psychiatric conditions, which display both alterations in polyamine levels and altered expression of genes related to polyamine metabolism. Studies have identified associations between genetic variants in spermidine/spermine N1-acetyltransferase (SAT1) and both anxiety and suicide, and several polymorphisms appear to play important roles in determining gene expression.

Methodology/Principal Findings

We genotyped 63 polymorphisms, spread across four polyaminergic genes (SAT1, spermine synthase (SMS), spermine oxidase (SMOX), and ornithine aminotransferase like-1 (OATL1)), in 1255 French-Canadian individuals who have been followed longitudinally for 22 years. We assessed univariate associations with anxiety, mood disorders, and attempted suicide, as assessed during early adulthood. We also investigated the involvement of gene-environment interactions in terms of childhood abuse, and assessed internalizing and externalizing symptoms as endophenotypes mediating these interactions. Overall, each gene was associated with at least one main outcome: anxiety (SAT1, SMS), mood disorders (SAT1, SMOX), and suicide attempts (SAT1, OATL1). Several SAT1 polymorphisms displayed disease-specific risk alleles, and polymorphisms in this gene were involved in gene-gene interactions with SMS to confer risk for anxiety disorders, as well as gene-environment interactions between childhood physical abuse and mood disorders. Externalizing behaviors demonstrated significant mediation with regards to the association between OATL1 and attempted suicide, however there was no evidence that externalizing or internalizing behaviors were appropriate endophenotypes to explain the associations with mood or anxiety disorders. Finally, childhood sexual abuse did not demonstrate mediating influences on any of our outcomes.

Conclusions/Significance

These results demonstrate that genetic variants in polyaminergic genes are associated with psychiatric conditions, each of which involves a set of separate and distinct risk alleles. As several of these polymorphisms are associated with gene expression, these findings may provide mechanisms to explain the alterations in polyamine metabolism which have been observed in psychiatric disorders.     

Identification of a chemical that inhibits the mycobacterial UvrABC complex in nucleotide excision repair

Bacterial DNA can be damaged by reactive nitrogen and oxygen intermediates (RNI and ROI) generated by host immunity, as well as by antibiotics that trigger bacterial production of ROI. Thus a pathogen's ability to repair its DNA may be important for persistent infection. A prominent role for nucleotide excision repair (NER) in disease caused by Mycobacterium tuberculosis (Mtb) was suggested by attenuation of uvrB-deficient Mtb in mice. However, it was unknown if Mtb's Uvr proteins could execute NER. Here we report that recombinant UvrA, UvrB, and UvrC from Mtb collectively bound and cleaved plasmid DNA exposed to ultraviolet (UV) irradiation or peroxynitrite. We used the DNA incision assay to test the mechanism of action of compounds identified in a high-throughput screen for their ability to delay recovery of M. smegmatis from UV irradiation. 2-(5-Amino-1,3,4-thiadiazol-2-ylbenzo[f]chromen-3-one) (ATBC) but not several closely related compounds inhibited cleavage of damaged DNA by UvrA, UvrB, and UvrC without intercalating in DNA and impaired recovery of M. smegmatis from UV irradiation. ATBC did not affect bacterial growth in the absence of UV exposure, nor did it exacerbate the growth defect of UV-irradiated mycobacteria that lacked uvrB. Thus, ATBC appears to be a cell-penetrant, selective inhibitor of mycobacterial NER. Chemical inhibitors of NER may facilitate studies of the role of NER in prokaryotic pathobiology.     

Inhibition of fatty acid oxidation by etomoxir impairs NADPH production and increases reactive oxygen species resulting in ATP depletion and cell death in human glioblastoma cells

Normal differentiated cells rely primarily on mitochondrial oxidative phosphorylation to produce adenosine triphosphate (ATP) to maintain their viability and functions by using three major bioenergetic fuels: glucose, glutamine and fatty acids. Many cancer cells, however, rely on aerobic glycolysis for their growth and survival, and recent studies indicate that some cancer cells depend on glutamine as well. This altered metabolism in cancers occurs through oncogene activation or loss of tumor suppressor genes in multiple signaling pathways, including the phosphoinositide 3-kinase and Myc pathways. Relatively little is known, however, about the role of fatty acids as a bioenergetic fuel in growth and survival of cancer cells. Here, we report that human glioblastoma SF188 cells oxidize fatty acids and that inhibition of fatty acid β-oxidation by etomoxir, a carnitine palmitoyltransferase 1 inhibitor, markedly reduces cellular ATP levels and viability. We also found that inhibition of fatty acid oxidation controls the NADPH level. In the presence of reactive oxygen species scavenger tiron, however, ATP depletion is prevented without restoring fatty acid oxidation. This suggests that oxidative stress may lead to bioenergetic failure and cell death. Our work provides evidence that mitochondrial fatty acid oxidation may provide NADPH for defense against oxidative stress and prevent ATP loss and cell death.     

Genetic dissection of Al tolerance QTLs in the maize genome by high density SNP scan

Background

Aluminum (Al) toxicity is an important limitation to food security in tropical and subtropical regions. High Al saturation on acid soils limits root development, reducing water and nutrient uptake. In addition to naturally occurring acid soils, agricultural practices may decrease soil pH, leading to yield losses due to Al toxicity. Elucidating the genetic and molecular mechanisms underlying maize Al tolerance is expected to accelerate the development of Al-tolerant cultivars.

Results

Five genomic regions were significantly associated with Al tolerance, using 54,455 SNP markers in a recombinant inbred line population derived from Cateto Al237. Candidate genes co-localized with Al tolerance QTLs were further investigated. Near-isogenic lines (NILs) developed for ZmMATE2 were as Al-sensitive as the recurrent line, indicating that this candidate gene was not responsible for the Al tolerance QTL on chromosome 5, qALT5. However, ZmNrat1, a maize homolog to OsNrat1, which encodes an Al³⁺ specific transporter previously implicated in rice Al tolerance, was mapped at ~40 Mbp from qALT5. We demonstrate for the first time that ZmNrat1 is preferentially expressed in maize root tips and is up-regulated by Al, similarly to OsNrat1 in rice, suggesting a role of this gene in maize Al tolerance. The strongest-effect QTL was mapped on chromosome 6 (qALT6), within a 0.5 Mbp region where three copies of the Al tolerance gene, ZmMATE1, were found in tandem configuration. qALT6 was shown to increase Al tolerance in maize; the qALT6-NILs carrying three copies of ZmMATE1 exhibited a two-fold increase in Al tolerance, and higher expression of ZmMATE1 compared to the Al sensitive recurrent parent. Interestingly, a new source of Al tolerance via ZmMATE1 was identified in a Brazilian elite line that showed high expression of ZmMATE1 but carries a single copy of ZmMATE1.

Conclusions

High ZmMATE1 expression, controlled either by three copies of the target gene or by an unknown molecular mechanism, is responsible for Al tolerance mediated by qALT6. As Al tolerant alleles at qALT6 are rare in maize, marker-assisted introgression of this QTL is an important strategy to improve maize adaptation to acid soils worldwide.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-153) contains supplementary material, which is available to authorized users.     

Heterologous expression and characterization of a "Pseudomature" form of taxadiene synthase involved in paclitaxel (Taxol) biosynthesis and evaluation of a potential intermediate and inhibitors of the multistep diterpene cyclization reaction

The diterpene cyclase taxadiene synthase from yew (Taxus) species transforms geranylgeranyl diphosphate to taxa-4(5),11(12)-diene as the first committed step in the biosynthesis of the anti-cancer drug Taxol. Taxadiene synthase is translated as a preprotein bearing an N-terminal targeting sequence for localization to and processing in the plastids. Overexpression of the full-length preprotein in Escherichia coli and purification are compromised by host codon usage, inclusion body formation, and association with host chaperones, and the preprotein is catalytically impaired. Since the transit peptide-mature enzyme cleavage site could not be determined directly, a series of N-terminally truncated enzymes was created by expression of the corresponding cDNAs from a suitable vector, and each was purified and kinetically evaluated. Deletion of up to 79 residues yielded functional protein; however, deletion of 93 or more amino acids resulted in complete elimination of activity, implying a structural or catalytic role for the amino terminus. The pseudomature form of taxadiene synthase having 60 amino acids deleted from the preprotein was found to be superior with respect to level of expression, ease of purification, solubility, stability, and catalytic activity with kinetics comparable to the native enzyme. In addition to the major product, taxa-4(5),11(12)-diene (94%), this enzyme produces a small amount of the isomeric taxa-4(20), 11(12)-diene ( approximately 5%), and a product tentatively identified as verticillene ( approximately 1%). Isotopically sensitive branching experiments utilizing (4R)-[4-(2)H(1)]geranylgeranyl diphosphate confirmed that the two taxadiene isomers, and a third (taxa-3(4),11(12)-diene), are derived from the same intermediate taxenyl C4-carbocation. These results, along with the failure of the enzyme to utilize 2, 7-cyclogeranylgeranyl diphosphate as an alternate substrate, indicate that the reaction proceeds by initial ionization of the diphosphate ester and macrocyclization to the verticillyl intermediate, followed by a secondary cyclization to the taxenyl cation and deprotonation (i.e., formation of the A-ring prior to B/C-ring closure). Two potential mechanism-based inhibitors were tested with recombinant taxadiene synthase but neither provided time-dependent inactivation nor afforded more than modest competitive inhibition.     

Simple isolation of functional RNA from woody stems of gymnosperms

Stems of woody conifers contain high levels of polysaccharides and phenolic compounds that complicate the isolation of functional RNA from this highly lignified tissue. These difficulties were overcome by pulverizing the frozen tissue with a stainless-steel mortar and by effectively sequestering interfering phenolic compounds with vinylpyrrolidone polymers, thereby minimizing damage to nucleic acids during extraction. RNases were inhibited by aurintricarboxylic acid, and gelatinous polysaccharides were removed by inclusion of a 10% ethanol precipitation step. RNA was then isolated by precipitation with 33% isopropanol and ultracentrifugation onto a cushion of 5.7 M CsCl. Yields of 10 to 20 μg RNA/g FW were obtained from woody stems of several different gymnosperm species, including grand fir (Abies grandis), lodgepole pine (Pinus contorta), loblolly pine (Pinus taeda), Douglas fir (Pseudotsuga menziesii) and Pacific yew (Taxus brevifolia). The high quality of the RNA obtained was determined by UV-spectrophotometry, denaturing agarose gel electrophoresis, andin-vitro translation, and this material was used to construct cDNA libraries.     

Monoterpene cyclases: use of the noncyclizable substrate analog 6,7-dihydrogeranyl pyrophosphate to uncouple the isomerization step of the coupled isomerization-cyclization reaction

Enzymes from Salvia officinalis capable of catalyzing the isomerization and subsequent cyclization of geranyl pyrophosphate to the monoterpenes (+)-alpha-pinene and (+)-bornyl pyrophosphate were examined with the noncyclizable substrate analog 6,7-dihydrogeranyl pyrophosphate in an attempt to dissect the cryptic isomerization step from the normally coupled reaction sequence. The analog inhibited the cyclization of geranyl pyrophosphate and was itself catalytically active, affording acyclic terpene olefins and alcohols as products. The enzymatic products generated from 6,7-dihydrogeranyl pyrophosphate qualitatively resembled the solvolysis products of 6,7-dihydrolinalyl pyrophosphate, yet they constituted a far higher proportion of olefins, suggesting that enzymatic product formation occurs in an environment relatively inaccessible to water. Since the normal cyclization of geranyl pyrophosphate is considered to proceed via preliminary isomerization to the bound tertiary intermediate (3R)-linalyl pyrophosphate, the results suggest that the analog undergoes the normal pyrophosphate ionization-migration step, giving rise in this case to (3R)-6,7-dihydrolinalyl pyrophosphate which is reionized, and because the subsequent cyclizations are precluded, the resulting cation is either deprotonated or captured by water. In divalent metal ion requirement, pH optimum, and other characteristics, the enzymatic transformation of the analog resembles the normal monoterpene cyclase reaction.