Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis

Vitamin C (l-ascorbic acid; AsA) acts as a potent antioxidant and cellular reductant in plants and animals. AsA has long been known to have many critical physiological roles in plants, yet its biosynthesis is only currently being defined. A pathway for AsA biosynthesis that features GDP-mannose and l-galactose has recently been proposed for plants. We have isolated a collection of AsA-deficient mutants of Arabidopsis thaliana that are valuable tools for testing of an AsA biosynthetic pathway. The best-characterized of these mutants (vtc1) contains ≈25% of wild-type AsA and is defective in AsA biosynthesis. By using a combination of biochemical, molecular, and genetic techniques, we have demonstrated that the VTC1 locus encodes a GDP-mannose pyrophosphorylase (mannose-1-P guanyltransferase). This enzyme provides GDP-mannose, which is used for cell wall carbohydrate biosynthesis and protein glycosylation as well as for AsA biosynthesis. In addition to genetically defining the first locus involved in AsA biosynthesis, this work highlights the power of using traditional mutagenesis techniques coupled with the Arabidopsis Genome Initiative to rapidly clone physiologically important genes.

Determining divergence times with a protein clock: Update and reevaluation

A recent study of the divergence times of the major groups of organisms as gauged by amino acid sequence comparison has been expanded and the data have been reanalyzed with a distance measure that corrects for both constraints on amino acid interchange and variation in substitution rate at different sites. Beyond that, the availability of complete genome sequences for several eubacteria and an archaebacterium has had a great impact on the interpretation of certain aspects of the data. Thus, the majority of the archaebacterial sequences are not consistent with currently accepted views of the Tree of Life which cluster the archaebacteria with eukaryotes. Instead, they are either outliers or mixed in with eubacterial orthologs. The simplest resolution of the problem is to postulate that many of these sequences were carried into eukaryotes by early eubacterial endosymbionts about 2 billion years ago, only very shortly after or even coincident with the divergence of eukaryotes and archaebacteria. The strong resemblances of these same enzymes among the major eubacterial groups suggest that the cyanobacteria and Gram-positive and Gram-negative eubacteria also diverged at about this same time, whereas the much greater differences between archaebacterial and eubacterial sequences indicate these two groups may have diverged between 3 and 4 billion years ago.

MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration

Exposure of eukaryotic cells to extracellular stimuli results in activation of mitogen-activated protein kinase (MAPK) cascades composed of MAPKs, MAPK kinases (MAP2Ks), and MAPK kinase kinases (MAP3Ks). Mammals possess a large number of MAP3Ks, many of which can activate the c-Jun N-terminal kinase (JNK) MAPK cascade when overexpressed, but whose biological function is poorly understood. We examined the function of the MAP3K MEK kinase 1 (MEKK1) in proinflammatory signaling. Using MEKK1-deficient embryonic stem cells prepared by gene targeting, we find that, in addition to its function in JNK activation by growth factors, MEKK1 is required for JNK activation by diverse proinflammatory stimuli, including tumor necrosis factor α, IL-1, double-stranded RNA, and lipopolysaccharide. MEKK1 is also essential for induction of embryonic stem cell migration by serum factors, but is not required for activation of other MAPKs or the IκB kinase signaling cascade.

The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates, chorismate and glutamine, and a product, glutamate, and (ii) its end-product inhibitor, l-tryptophan

The crystal structure of anthranilate synthase (AS) from Serratia marcescens, a mesophilic bacterium, has been solved in the presence of its substrates, chorismate and glutamine, and one product, glutamate, at 1.95 Å, and with its bound feedback inhibitor, tryptophan, at 2.4 Å. In comparison with the AS structure from the hyperthermophile Sulfolobus solfataricus, the S. marcescens structure shows similar subunit structures but a markedly different oligomeric organization. One crystal form of the S. marcescens enzyme displays a bound pyruvate as well as a putative anthranilate (the nitrogen group is ambiguous) in the TrpE subunit. It also confirms the presence of a covalently bound glutamyl thioester intermediate in the TrpG subunit. The tryptophan-bound form reveals that the inhibitor binds at a site distinct from that of the substrate, chorismate. Bound tryptophan appears to prevent chorismate binding by a demonstrable conformational effect, and the structure reveals how occupancy of only one of the two feedback inhibition sites can immobilize the catalytic activity of both TrpE subunits. The presence of effectors in the structure provides a view of the locations of some of the amino acid residues in the active sites. Our findings are discussed in terms of the previously described AS structure of S. solfataricus, mutational data obtained from enteric bacteria, and the enzyme's mechanism of action.

Genomic analysis of orthologous mouse and human olfactory receptor loci

Olfactory receptor (OR) genes represent ≈1% of genomic coding sequence in mammals, and these genes are clustered on multiple chromosomes in both the mouse and human genomes. We have taken a comparative genomics approach to identify features that may be involved in the dynamic evolution of this gene family and in the transcriptional control that results in a single OR gene expressed per olfactory neuron. We sequenced ≈350 kb of the murine P2 OR cluster and used synteny, gene linkage, and phylogenetic analysis to identify and sequence ≈111 kb of an orthologous cluster in the human genome. In total, 18 mouse and 8 human OR genes were identified, including 7 orthologs that appear to be functional in both species. Noncoding homology is evident between orthologs and generally is confined within the transcriptional unit. We find no evidence for common regulatory features shared among paralogs, and promoter regions generally do not contain strong promoter motifs. We discuss these observations, as well as OR clustering, in the context of evolutionary expansion and transcriptional regulation of OR repertoires.