BackgroundPrincipal component analysis (PCA) has been widely used to visualize high-dimensional metabolomic data in a two- or three-dimensional subspace. In metabolomics, some metabolites (e.g., the top 10 metabolites) have been subjectively selected when using factor loading in PCA, and biological inferences are made for these metabolites. However, this approach may lead to biased biological inferences because these metabolites are not objectively selected with statistical criteria.ResultsWe propose a statistical procedure that selects metabolites with statistical hypothesis testing of the factor loading in PCA and makes biological inferences about these significant metabolites with a metabolite set enrichment analysis (MSEA). This procedure depends on the fact that the eigenvector in PCA for autoscaled data is proportional to the correlation coefficient between the PC score and each metabolite level. We applied this approach to two sets of metabolomic data from mouse liver samples: 136 of 282 metabolites in the first case study and 66 of 275 metabolites in the second case study were statistically significant. This result suggests that to set the number of metabolites before the analysis is inappropriate because the number of significant metabolites differs in each study when factor loading is used in PCA. Moreover, when an MSEA of these significant metabolites was performed, significant metabolic pathways were detected, which were acceptable in terms of previous biological knowledge.ConclusionsIt is essential to select metabolites statistically to make unbiased biological inferences from metabolomic data when using factor loading in PCA. We propose a statistical procedure to select metabolites with statistical hypothesis testing of the factor loading in PCA, and to draw biological inferences about these significant metabolites with MSEA. We have developed an R package “mseapca” to facilitate this approach. The “mseapca” package is publicly available at the CRAN website.

A novel endo-exonuclease, DmGEN (Drosophila Melanogaster XPG-like endonuclease), was identified in D.melanogaster. DmGEN is composed of five exons and four introns, and the open reading frame encodes a predicted product of 726 amino acid residues with a molecular weight of 82.5 kDa and a pI of 5.36. The gene locus on Drosophila polytene chromosomes was detected at 64C9 on the left arm of chromosome 3 as a single site. The encoded protein showed a relatively high degree of sequence homology with the RAD2 nucleases, especially XPG. Although the XPG-N- and XPG-I-domains are highly conserved in sequence, locations of the domains are similar to those of FEN-1 and EXO-1, and the molecular weight of the protein is close to that of EXO-1. In vitro, DmGEN showed endonuclease and 3'-5' exonuclease activities with both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), but the endonuclease action with dsDNA was quite specific: 5'-3' exonuclease activity was found to occur with nicked DNA, while dsDNA was endonucleolytically cut at 3-4 bp from the 5' end. Homologs are widely found in mammals and higher plants. The data suggest that DmGEN belongs to a new class of RAD2 nuclease.

Studies on the biochemical properties of very-large-size eukaryotic DNA polymerases have been limited by the difficulty in obtaining sufficient purified forms of each enzyme. Our aim was to determine and elucidate the biochemical properties of one such polymerase, pol zeta (DNA polymerase zeta) from Drosophila melanogaster (Dmpol zeta). Using an REV1 (UV-revertible gene 1) protein-affinity column, we have isolated the enzyme directly from Drosophila embryos. Completely purified Dmpol zeta was found to have a molecular mass of approx. 240 kDa, and to be sensitive to aphidicolin and resistant to ddTTP (2',3'-dideoxythymidine-5-triphosphate) and N-ethylmaleimide. The enzyme has a preference for poly(dA)/oligo(dT)(10:1) as a template primer and has high processivity for DNA synthesis. Moreover, Dmpol zeta showed significantly higher fidelity compared with Rattus norvegicus DNA polymerase, an error-prone DNA polymerase, in an M13 forward mutation assay. The activities of bypassing pyrimidine dimers and (6-4) photoproducts and extending from mismatched primer-template termini in (6-4) photoproduct by Dmpol zeta were not detected. Drosophila REV7 interacted with Dmpol zeta in vitro, but did not influence the DNA synthesis activity of Dmpol zeta. The present study is the first report about characterization of purified pol zeta from multicellular organisms, and the second concerning the characterization of yeast pol zeta.

DNA replication, recombination and repair are key processes in maintaining genome integrity. Nucleases are necessary for their nucleolytic activities. They act on a variety of structural frameworks, ranging from sitespecific (e.g. AP endonuclease) to structure-specific (e.g. RAD2 ⁄ XPG nuclease family) and nonspecific (e.g. DNase I) nucleases. In particular, members of the RAD2 ⁄ XPG nuclease family have unique nuclease activities and play critical roles in genome stability [1][2][3][4][5][6]. In a preliminary report, we described the presence of a new nuclease, Drosophila melanogaster XPG-like endonuclease (DmGEN) which belongs to the RAD2 ⁄ XPG nuclease family, shows unique activity and possibly plays a critical role in genome stability [7]. The ORF of the DmGEN gene encoded a predicted protein of 726 amino acid residues with a molecular mass of 82.5 kDa. The gene was located at 64C9 on the left arm of Drosophila polytene chromosome 3 as a single site.The RAD2 ⁄ XPG family of nucleases, which have two conserved nuclease domains (the N domain and the I domain), are currently separated into three classes (XPG ⁄ class I, FEN-1 ⁄ class II and EXO-1 ⁄ class III) based on the types of nuclease activity and sequence homology [8,9]. In Drosophila, mus201 protein (class I), FEN-1 homologue protein (class II), and Tosca protein (class III) have been reported as RAD2 family proteins. The DmGEN protein showed a relatively high degree of sequence homology with RAD2 nucleases, particularly XPG, although the locations of the N and I domains were similar to those of FEN-1 and EXO-1, and the molecular mass of DmGEN was found to be close to that of EXO-1. Therefore, we proposed a new class (class IV) to categorize DmGEN and SEND-1, which we also found in higher plants [8]. Recently, a new member of the class IV nucleases, OsGEN-like, has been reported in rice; RNA-mediated Drosophila melanogaster XPG-like endonuclease (DmGEN) is a new category of nuclease belonging to the RAD2 ⁄ XPG family. The DmGEN protein has two nuclease domains (N and I domains) similar to XPG ⁄ class I nucleases; however, unlike class I nucleases, in DmGEN these two nuclease domains are positioned close to each other as in FEN-1 ⁄ class II and EXO-1 ⁄ class III nucleases. To confirm the properties of DmGEN, we characterized the active-site mutant protein (E143A E145A) and found that DmGEN had flap endonuclease activity. DmGEN possessed weak nick-dependent 5¢)3¢ exonuclease activity. Unlike XPG, DmGEN could not incise the bubble structure. Interestingly, based on characterization of flap endonuclease activity, DmGEN preferred the blocked-flap structure as a substrate. This feature is distinctly different from FEN-1. Furthermore, DmGEN cleaved the lagging strand of the model replication fork. Immunostaining revealed that DmGEN was present in the nucleus of actively proliferating Drosophila embryos. Thus, our studies revealed that DmGEN belongs to a new class (class IV) of the RAD2 ⁄ XPG nuclease family. The biochemical properties of DmGEN and its possible r...

We describe genetically proven monozygotic twins with the adult form of adrenoleukodystrophy with significant phenotypic heterogeneity. Myeloneuropathy was common to both patients, but cognitive impairment and affective symptoms with extensive demyelination in the brain were prominent in the older twin, while adrenal insufficiency was predominant in the younger twin. The younger twin, however, exhibited affective symptoms similar to those displayed by his elder twin 10 years later. These findings suggest that nongenetic factors are important in determining the phenotypic variation of adrenoleukodystrophy gene.