All pyruvylated galactose in Schizosaccharomyces pombe N-glycans is present in the terminal disaccharide, 4, 6-O-[(R)-(1- carboxyethylidine)]-Galbeta1,3Galalpha1-

The large N-linked oligosaccharides released from Schizosaccharomyces pombe by endo-beta-N-acetylglucosaminidase H were examined to determine how the negatively chargedpyruvylated galactoses present (Gemmill,T.R., and Trimble,R.B., 1996, J. Biol. Chem ., 271, 25945-25949) were attached to the oligosaccharide chains. Binding of biotinylated human serum amyloid P and peanut agglutinin to native and depyruvylated S.pombe glycoproteins, respectively, indicated that the pyruvylated epitope was likely to be in the beta configuration. Examination by high- field 1H NMR of whole glycans and a disaccharide fragment released from them on partial acid hydrolysis showed that the pyruvylated galactose species was in fact beta1,3-linked to a second galactose, and this occurred an average of five to six times on nominal Gal57Man64GlcNAc N- glycans. The pyruvate-2,(4,6)Gal-beta1,3Gal epitope is chemically similar to acetaldehyde-Galbeta1,3Gal groups found on the glycoproteins from Paramyxovirus-infected bovine kidney cells (Prehm, P., Scheid,A. and Choppin,P.W. ,1979, J. Biol. Chem ., 254, 9669-9677). The 1:1 stoichiometry between pyruvate and beta-linked galactose in these S.pombe glycans indicates that either pyruvate addition to terminal beta1,3Gal is highly efficient or that pyruvylated Gal is transferred en bloc to alpha1,2-linked Gal residues in theN-linked chains. In contradiction to many galactomannan-producing fungi, which add substantial amounts of Gal in the furanose form to their glycoproteins, all detectable Gal in the large S.pombe galactomannans is in the pyranose form, as found in higher eukaryotes. The current work shows that the S.pombe outer chain structure is a poly-alpha1,6Man backbone 2- O-substituted with either Gal or the pyruvylated galactobiose and contains little alpha1,2-linked or 2-O-substituted Man. This is in contrast to the S. cerevisiae outer chain, which is poly-alpha1,6Man substituted with alpha1,2-linked Man sidechains (Ballou,C.E. ,1990, Methods Enzymol , 185, 440-470).      

Lotus corniculatus nodulation specificity is changed by the presence of a soybean lectin gene

Plant lectins have been implicated as playing an important role in mediating recognition and specificity in the Rhizobium-legume nitrogen-fixing symbiosis. To test this hypothesis, we introduced the soybean lectin gene Le1 either behind its own promoter or behind the cauliflower mosaic virus 35S promoter into Lotus corniculatus, which is nodulated by R. loti. We found that nodulelike outgrowths developed on transgenic L. corniculatus plant roots in response to Bradyrhizobium japonicum, which nodulates soybean and not Lotus spp. Soybean lectin was properly targeted to L. corniculatus root hairs, and although infection threads formed, they aborted in epidermal or hypodermal cells. Mutation of the lectin sugar binding site abolished infection thread formation and nodulation. Incubation of bradyrhizobia in the nodulation (nod) gene-inducing flavonoid genistein increased the number of nodulelike outgrowths on transgenic L. corniculatus roots. Studies of bacterial mutants, however, suggest that a component of the exopolysaccharide surface of B. japonicum, rather than Nod factor, is required for extension of host range to the transgenic L. corniculatus plants.     

High-throughput protein purification and quality assessment for crystallization

The ultimate goal of structural biology is to understand the structural basis of proteins in cellular processes. In structural biology, the most critical issue is the availability of high-quality samples. "Structural biology-grade" proteins must be generated in the quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. The purification procedures must reproducibly yield homogeneous proteins or their derivatives containing marker atom(s) in milligram quantities. The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. With structural genomics emphasizing a genome-based approach in understanding protein structure and function, a number of unique structures covering most of the protein folding space have been determined and new technologies with high efficiency have been developed. At the Midwest Center for Structural Genomics (MCSG), we have developed semi-automated protocols for high-throughput parallel protein expression and purification. A protein, expressed as a fusion with a cleavable affinity tag, is purified in two consecutive immobilized metal affinity chromatography (IMAC) steps: (i) the first step is an IMAC coupled with buffer-exchange, or size exclusion chromatography (IMAC-I), followed by the cleavage of the affinity tag using the highly specific Tobacco Etch Virus (TEV) protease; the second step is IMAC and buffer exchange (IMAC-II) to remove the cleaved tag and tagged TEV protease. These protocols have been implemented on multidimensional chromatography workstations and, as we have shown, many proteins can be successfully produced in large-scale. All methods and protocols used for purification, some developed by MCSG, others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Diseases (CSGID) purification pipeline, are discussed in this chapter.     

Comparisons between Geographically Diverse Samples of Carried Staphylococcus aureus

Approximately one-third of the human population is asymptomatically colonized by Staphylococcus aureus. However, much of the global diversity within the carriage populations remains uncharacterized, and it is unclear to what degree the variation is geographically partitioned. We isolated 300 carriage isolates from 1,531 adults contemporaneously in four countries: France, Algeria, Moldova, and Cambodia. All strains were characterized by multilocus sequence typing. Six clonal complexes (CCs) were present in all four samples (CC30, -45, -121, -15, -5, and -8). Analyses based on the genotype frequencies revealed the French and Algerian samples to be most similar and the Cambodian sample to be most distinct. While this pattern is consistent with likely rates of human migration and geographic distance, stochastic clonal expansion also contributes to regional differences. Phylogenetic analysis revealed a highly divergent and uncharacterized genotype (ST1223) within Cambodia. This lineage is related to CC75, which has previously been observed only in remote aboriginal populations in northern Australia.Although better known as an important human pathogen, Staphylococcus aureus is typically a commensal species and asymptomatically colonizes approximately one-third of the human population globally (18, 20, 29). This high carriage rate potentially represents a vast reservoir of as-yet-uncharacterized S. aureus diversity, an appreciation of which should shed light on the forces underpinning the diversification and dissemination of S. aureus. There are comparatively few studies examining spatial or temporal genotype distributions within carriage populations, and the extent of biogeographical structure is currently unclear, as is the level of discrimination which might be required to detect such structure.Multilocus sequence typing (MLST) has proved to be very successful as an epidemiological tool in that it delimits S. aureus in to a small number of widespread and discrete clonal complexes (CCs) (6, 8). These can be readily identified as clusters of related genotypes which have diversified radially from “founder” genotypes (9), and because this organism is largely clonal (8), assignments of isolates to these groups is broadly robust to the many different typing methods employed (4, 10, 27). The high level of divergence between these lineages suggests that they are relatively ancient and temporally stable (7), and it is possible that isolated host populations may have been colonized by different S. aureus lineages in the past. However, any footprints of geographical partitioning are likely to have been compromised by high rates of migration in recent times, due largely to the advent of air travel.Previous studies addressing the characterization of carried populations have tended to focus on samples from Western Europe or North America, and these have generally not provided strong evidence for geographical structuring. In a recent study using amplified fragment length polymorphism to compare the carried populations in Holland and North America, the authors noted considerable overlap between the samples, suggesting that they effectively constituted a single unstructured population (17). Similarly, independent MLST studies have revealed regional consistencies in Europe, such as the predominance of CC30 in the United Kingdom (8), Ireland (3), and Switzerland (25). Given the high rates of admixture within Europe and North America, the absence of obvious geographical structuring in the carried S. aureus population in these regions is perhaps not very surprising.Although they are currently scarce, current data from carriage populations outside of Europe or North America point to greater geographical structuring. For example, a sample of carried S. aureus recovered from Bamako, Mali, has recently been characterized, constituting the first such study of an African population (23). Although many of the previously characterized CCs were also present in this sample, the authors noted a high frequency (∼25%) of a single genotype, ST152, which is phylogenetically divergent and noted very rarely in Europe. The high frequency of ST152 in this population raises the possibility that this genotype is endemic to the Malian population and possibly elsewhere in sub-Saharan Africa. This in turn hints at greater geographical partitioning on a global scale, although more representative samples are clearly required. To address this, we generated MLST data from contemporaneous carriage samples recovered from four countries representing three continents: France (Western Europe), Moldova (Eastern Europe), Algeria (North Africa), and Cambodia (Southeast Asia). To our knowledge, this is the first time such a study has been carried out on samples from Eastern Europe, North Africa, or Southeast Asia. These data were therefore generated to uncover diversity within the global carriage population but also to understand further the extent to which geographical distance and host migration can explain regional differences.     

An RNA folding method capable of identifying pseudoknots and base triples

MOTIVATION: Recently, we described a Maximum Weighted Matching (MWM) method for RNA structure prediction. The MWM method is capable of detecting pseudoknots and other tertiary base-pairing interactions in a computationally efficient manner (Cary and Stormo, Proceedings of the Third International Conference on Intelligent Systems for Molecular Biology, pp. 75-80, 1995). Here we report on the results of our efforts to improve the MWM method's predictive accuracy, and show how the method can be extended to detect base interactions formerly inaccessible to automated RNA modeling techniques. RESULTS: Improved performance in MWM structure prediction was achieved in two ways. First, new ways of calculating base pair likelihoods have been developed. These allow experimental data and combined statistical and thermodynamic information to be used by the program. Second, accuracy was improved by developing techniques for filtering out spurious base pairs predicted by the MWM program. We also demonstrate here a means by which the MWM folding method may be used to detect the presence of base triples in RNAs. AVAILABILITY: http://www.cshl.org/mzhanglab/tabaska/j axpage. html CONTACT: tabaska@cshl.org      

Cloning and gene map assignment of the Xiphophorus DNA ligase 1 gene

Fishes represent the stem vertebrate condition and have maintained several gene arrangements common to mammalian genomes throughout the 450 Myr of divergence from a common ancestor. One such syntenic arrangement includes the GPI-PEPD enzyme association on Xiphophorus linkage group IV and human chromosome 19. Previously we assigned the Xiphophorus homologue of the human ERCC2 gene to linkage group U5 in tight association with the CKM locus. CKM is also tightly linked to the ERCC2 locus on human chromosome 19, leading to speculation that human chromosome 19 may have arisen by fusion of two ancestral linkage groups which have been maintained in fishes. To investigate this hypothesis further, we isolated and sequenced Xiphophorus fish genomic regions exhibiting considerable sequence similarity to the human DNA ligase 1 amino acid sequence. Comparison of the fish DNA ligase sequence with those of other species suggests several modes of amino acid conservation in this gene. A 2.2-kb restriction fragment containing part of an X. maculatus DNA ligase 1 exon was used in backcross hybrid mapping with 12 enzyme or RFLP loci. Significant linkage was observed between the nucleoside phosphorylase (NP2) and the DNA ligase (LIG1) loci on Xiphophorus linkage group VI. This assignment suggests that the association of four DNA repair-related genes on human chromosome 19 may be the result of chance chromosomal rearrangements.