Alteration in sphingolipid metabolism: bioassays for fumonisin- and ISP-I-like activity in tissues, cells and other matrices

The first discovered naturally occurring inhibitor of de novo sphingolipid biosynthesis was fumonisin B1. There are now 11 identified fungal inhibitors of ceramide synthase or 'fumonisin B1-like' compounds. With the exception of the australifungins, all other fungal ceramide synthase inhibitors are structurally sphingoid-like. There are several recently discovered fungal inhibitors of another enzyme in the de novo sphingolipid biosynthesis pathway: serine palmitoyltransferase (SPT). One of the SPT inhibitors is named ISP-I. While ceramide synthase inhibitors are toxic to animals, plants and fungi, the SPT inhibitors are not known to cause animal or plant disease, but are potent inhibitors of fungal growth. Very little is known about their toxicity in animals. There are at least 24 fungal SPT inhibitors produced by a variety of fungi. Given that the fungal inhibitors of sphingolipid biosynthesis are chemically and biologically diverse, two bioassays have been developed to screen for fumonisin-like or ISP-I-like activity in naturally contaminated products or fungal culture materials. These bioassays are based on the changes in free sphingoid base concentration that occur when the ceramide synthase or SPT are inhibited. The bioassays have the advantage that they are functionally rather than chemically specific and thus will detect ceramide synthase and SPT inhibitors regardless of their chemical structure.     

The residues between the two transformation effector sites of Epstein-Barr virus latent membrane protein 1 are not critical for B-lymphocyte growth transformation

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is essential for EBV-mediated transformation of primary B lymphocytes. LMP1 spontaneously aggregates in the plasma membrane and enables two transformation effector sites (TES1 and TES2) within the 200-amino-acid cytoplasmic carboxyl terminus to constitutively engage the tumor necrosis factor receptor (TNFR)-associated factors TRAF1, TRAF2, TRAF3, and TRAF5 and the TNFR-associated death domain proteins TRADD and RIP, thereby activating NF-kappaB and c-Jun N-terminal kinase (JNK). To investigate the importance of the 60% of the LMP1 carboxyl terminus that lies between the TES1-TRAF and TES2-TRADD and -RIP binding sites, an EBV recombinant was made that contains a specific deletion of LMP1 codons 232 to 351. Surprisingly, the deletion mutant was similar to wild-type (wt) LMP1 EBV recombinants in its efficiency in transforming primary B lymphocytes into lymphoblastoid cell lines (LCLs). Mutant and wt EBV-transformed LCLs were similarly efficient in long-term outgrowth and in regrowth after endpoint dilution. Mutant and wt LMP1 proteins were also similar in their constitutive association with TRAF1, TRAF2, TRAF3, TRADD, and RIP. Mutant and wt EBV-transformed LCLs were similar in steady-state levels of Bcl2, JNK, and activated JNK proteins. The wt phenotype of recombinants with LMP1 codons 232 to 351 deleted further demarcates TES1 and TES2, underscores their central importance in B-lymphocyte growth transformation, and provides a new perspective on LMP1 sequence variation between TES1 and TES2.     

Short tandem repeats are associated with diverse mRNAs encoding membrane-targeted proteins

Within the genomes of multicellular organisms, short tandem repeating sequences (STRs) are ubiquitous, yet usage patterns remain obscure. The repeats (AC)n and (GU)n appear frequently in the untranslated regions (UTRs) of messenger RNAs (mRNAs). To investigate STR usage patterns, we used three approaches: (1) comparisons of individual mRNA database sequences including annotations and linked references, (2) statistical analysis of complete, UTR databases and (3) study of a large gene family, the aquaporins. Among 500 (AC)n- or (GU)n-containing mRNAs, 58 (12%) had known functions. Of these, 50 (86%) encoded proteins whose activities involved membranes or lipids, including integral membrane proteins, peripheral membrane proteins, ion channels, lipid enzymes, receptors and secreted proteins. A control sequence (AU)n also occurred in mRNAs, but only 5% encoded membrane-related functions. Investigation of all reported 3' UTR sequences, demonstrated that the STR (AC)n was 9 times more common in mRNAs encoding membrane functions than in the total UTR database (P < 0.001). Similarly, (GU)n was 8 times more common in membrane-function mRNAs than in the total database (P < 0.001). These observations suggest that (AC)n and (GU)n may be UTR signals for some mRNAs encoding membrane-targeted proteins.     

Structural domains, protein modules, and sequence similarities enrich our understanding of the Shewanella oneidensis MR-1 proteome

The protein coding sequences of S. oneidensis MR-1 were analyzed, and new annotations were given to 491 gene products, 306 of which were previously of unknown function. New information was mainly brought in from structural domain predictions for S. oneidensis proteins of the SUPERFAM database (http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY/) and newly identified and experimentally verified functions of homologous proteins. Proteins encoded by fused genes were identified and separated into modules, protein units of at least 83 aa with independent functions and distinct evolutionary histories. A reannotation of the fused gene products was done to assign functions to the appropriate module within the protein. Groups of sequence-similar proteins of S. oneidensis were assembled. The fused gene products were represented by their modular entities for the grouping process. The protein groups were analyzed for their size and functions, and they were used to indicate activities that are of importance to the environmental adaptation of this organism. Making use of several approaches not commonly used in annotation, we have been able to enrich our understanding of the functions encoded by the S. oneidensis genome.     

A molecular phylogeny of enteric bacteria and implications for a bacterial species concept

A molecular phylogeny for seven taxa of enteric bacteria (Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae, and Serratia plymuthica) was made from multiple isolates per taxa taken from a collection of environmental enteric bacteria. Sequences from five housekeeping genes (gapA, groEL, gyrA, ompA, and pgi) and the 16S rRNA gene were used to infer individual gene trees and were concatenated to infer a composite molecular phylogeny for the species. The isolates from each taxa formed tight species clusters in the individual gene trees, suggesting the existence of 'genotypic' clusters that correspond to traditional species designations. These sequence data and the resulting gene trees and consensus tree provide the first data set with which to assess the utility of the recently proposed core genome hypothesis (CGH). The CGH provides a genetically based approach to applying the biological species concept to bacteria.     

A functional update of the Escherichia coli K-12 genome

Background  

Since the genome of Escherichia coli K-12 was initially annotated in 1997, additional functional information based on biological characterization and functions of sequence-similar proteins has become available. On the basis of this new information, an updated version of the annotated chromosome has been generated.     

Insertional tagging of regulatory sequences in tritordeum; a hexaploid cereal species

As an approach to isolate novel cereal promoters, promoterless uidA constructs and particle bombardment were used to transform tritordeum. Five of eight transgenic lines containing uidA sequences showed evidence of promoter tagging. Expression of uidA was detected in four lines as: constitutive expression, expression in short cells of the epidermis of the spikelets, expression in pollen grains and in cells of the epidermis of the spikelet, and expression in anther primordia and pollen grains. In the fifth line, the uidA was shown by RT-PCR to be transcribed, but no GUS activity was detected. The different patterns of uidA expression indicate that different regulatory sequences were tagged in each of these lines. Analysis of the progeny resulting from self-fertilisation of the primary tagged plants, indicate that the transgenes integrated at one or two loci and the patterns of expression were stably inherited. To our knowledge, this is the first report of promoter tagging in cereals by direct gene transfer.