Adventitious roots inGlycine subg.Glycine (Leguminosae): Morphological and taxonomic indicators of the B genome

Adventitious roots were observed on 3 wild perennialGlycine species, in short-poddedG. clandestina, G. latifolia and mostG. tabacina (2x, 4x, 6x), while other species lacked adventitious roots. This suggests that the adventitious roots trait is associated with B genome species. Intra- and interspecific F₁ hybrids reveal that adventitious roots apparently are inherited recessively. The presence of adventitious roots or short-poddedG. clandestina, coupled with infertility with longer-poddedG. clandestina, and enzymatic and leaflet morphology differences between the twoG. clandestina subgroups, supports segregation of the short-podded form as a separate taxon.     

The crystal structure of the costimulatory OX40-OX40L complex

OX40 is a T cell costimulator activated by OX40L. Blockade of the OX40L-OX40 interaction has ameliorative effects in animal models of T cell pathologies. In order to better understand the interaction between OX40 and OX40L, we have determined the crystal structure of murine OX40L and of the human OX40-OX40L complex at 1.45 and 2.4 A, respectively. These structures show that OX40L is an unusually small member of the tumor necrosis factor superfamily (TNFSF). The arrangement of the OX40L protomers forming the functional trimer is atypical and differs from that of other members by a 15 degrees rotation of each protomer with respect to the trimer axis, resulting in an open assembly. Site-directed changes of the interfacial residues of OX40L suggest this interface lacks a single "hot spot" and that instead, binding energy is dispersed over at least two distinct areas. These structures demonstrate the structural plasticity of TNFSF members and their interactions with receptors.     

Ubiquitin binding to A20 ZnF4 is required for modulation of NF-κB signaling

Inactivating mutations in the ubiquitin (Ub) editing protein A20 promote persistent nuclear factor (NF)-κB signaling and are genetically linked to inflammatory diseases and hematologic cancers. A20 tightly regulates NF-κB signaling by acting as an Ub editor, removing K63-linked Ub chains and mediating addition of Ub chains that target substrates for degradation. However, a precise molecular understanding of how A20 modulates this pathway remains elusive. Here, using structural analysis, domain mapping, and functional assays, we show that A20 zinc finger?4 (ZnF4) does not directly interact with E2 enzymes but instead can bind mono-Ub and K63-linked poly-Ub. Mutations to the A20 ZnF4 Ub-binding surface result in decreased A20-mediated ubiquitination and impaired regulation of NF-κB signaling. Collectively, our studies illuminate the mechanistically distinct but biologically interdependent activities of the A20 ZnF and ovarian tumor (OTU) domains that are inherent to the Ub editing process and, ultimately, to regulation of NF-κB signaling.     

Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

Proper hedgehog (Hh) signaling is crucial for embryogenesis and tissue regeneration. Dysregulation of this pathway is associated with several types of cancer. The monoclonal antibody 5E1 is a Hh pathway inhibitor that has been extensively used to elucidate vertebrate Hh biology due to its ability to block binding of the three mammalian Hh homologs to the receptor, Patched1 (Ptc1). Here, we engineered a murine:human chimeric 5E1 (ch5E1) with similar Hh-binding properties to the original murine antibody. Using biochemical, biophysical, and x-ray crystallographic studies, we show that, like the regulatory receptors Cdon and Hedgehog-interacting protein (Hhip), ch5E1 binding to Sonic hedgehog (Shh) is enhanced by calcium ions. In the presence of calcium and zinc ions, the ch5E1 binding affinity increases 10–20-fold to tighter than 1 nm primarily because of a decrease in the dissociation rate. The co-crystal structure of Shh bound to the Fab fragment of ch5E1 reveals that 5E1 binds at the pseudo-active site groove of Shh with an epitope that largely overlaps with the binding site of its natural receptor antagonist Hhip. Unlike Hhip, the side chains of 5E1 do not directly coordinate the Zn²⁺ cation in the pseudo-active site, despite the modest zinc-dependent increase in 5E1 affinity for Shh. Furthermore, to our knowledge, the ch5E1 Fab-Shh complex represents the first structure of an inhibitor antibody bound to a metalloprotease fold.     

SSR marker and ITS cleaved amplified polymorphic sequence analysis of soybean × Glycine tomentella intersubgeneric derived lines

Wild perennial Glycine species are an invaluable gene resource for the cultivated soybean [Glycine max (L.) Merr., 2n=40]. However, these wild species have been largely unexplored in soybean breeding programs because of their extremely low crossability with soybean and the need to employ in vitro embryo rescue methods to produce F₁ hybrids. The objective of this study was to develop molecular markers to identify gene introgression from G. tomentella, a wild perennial Glycine species, to soybean. A selection of 96 soybean simple sequence repeat (SSR) markers was evaluated for cross-specific amplification and polymorphism in G. tomentella. Thirty-two SSR markers (33%) revealed specific alleles for G. tomentella PI 483218 (2n=78). These SSR markers were further examined with an amphidiploid line (2n=118) and monosomic alien addition lines (MAALs), each with 2n=40 chromosomes from soybean and one from G. tomentella. The results show that the use of SSR markers is a rapid and reliable method to detect G. tomentella chromosomes in MAALs. We also developed a cleaved amplification polymorphism sequence (CAPS) marker according to the sequences of internal transcribed spacer (ITS) regions in soybean and G. tomentella. Four MAALs that carry the ITS (rDNA) locus from G. tomentella were identified. The SSR and ITS-CAPS markers will greatly facilitate the introgression and characterization of gene transfer from G. tomentella to soybean.Communicated by F.J. Muehlbauer     

A unique zinc-binding site revealed by a high-resolution X-ray structure of homotrimeric Apo2L/TRAIL

Apoptosis-inducing ligand 2 (Apo2L, also called TRAIL), a member of the tumor necrosis factor (TNF) family, induces apoptosis in a variety of human tumor cell lines but not in normal cells [Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C.-P., Nicholl, J. K., Sutherland, G. R., Smith, T. D., Rauch, C., Smith, C. A., and Goodwin, R. G. (1995) Immunity 3, 673-682; Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J., Moore, A., and Ashkenazi, A. (1996) J. Biol. Chem. 271, 12687-12690]. Here we describe the structure of Apo2L at 1.3 A resolution and use alanine-scanning mutagenesis to map the receptor contact regions. The structure reveals a homotrimeric protein that resembles TNF with receptor-binding epitopes at the interface between monomers. A zinc ion is buried at the trimer interface, coordinated by the single cysteine residue of each monomer. The zinc ion is required for maintaining the native structure and stability and, hence, the biological activity of Apo2L. This is the first example of metal-dependent oligomerization and function of a cytokine.     

Molecular recognition by a binary code

Functional antibodies were obtained from a library of antigen-binding sites generated by a binary code restricted to tyrosine and serine. An antibody raised against human vascular endothelial growth factor recognized the antigen with high affinity (K(D)=60 nM) and high specificity in cell-based assays. The crystal structure of another antigen binding fragment in complex with its antigen (human death receptor DR5) revealed the structural basis for this minimalist mode of molecular recognition. Natural antigen-binding sites are enriched for tyrosine and serine, and we show that these amino acid residues are intrinsically well suited for molecular recognition. Furthermore, these results demonstrate that molecular recognition can evolve from even the simplest chemical diversity.     

A molecular phylogenetic study of the subtribe Glycininae (Leguminosae) derived from the chloroplast DNA rps16 intron sequences

Phylogenetic relationships among 13 genera of the subtribe Glycininae, two genera of the allied subtribe Diocleinae that were included within Glycininae by Polhill, and two genera of the subtribe Erythrininae as outgroups were inferred from chloroplast DNA rps16 intron sequence variation. Pairwise sequence divergence values ranged from identity between Teramnus mollis and T. micans and between T. flexilis and T. labialis to 7.89% between Pueraria wallichii and Pseudeminia comosa across all accessions. Phylogenies estimated using parsimony and neighbor-joining methods revealed that (1) Glycininae is monophyletic if Pachyrhizus and Calopogonium (both Diocleinae) are included within Glycininae; (2) the genus Teramnus is closely related to Glycine, and Amphicarpaea showed a sister relationship to the clade comprising Teramnus and Glycine; (3) the expanded Glycininae including two genera of Diocleinae is divided into three branches, temporarily named I (comprising the rest of the examined taxa), II (Pueraria wallichii), and III (Mastersia), but their relationships are equivocal; and (4) the genus Pueraria, regarded as a closely related genus to Glycine, is not monophyletic and should be divided into at least four genera (a hypothesis supported previously by Lackey).     

Genomic diversity in aneudiploid (2n = 38) and diploid (2n = 40) Glycine tomentella revealed by cytogenetic and biochemical methods.

Of the 15 perennial species of the subgenus Glycine Willd., G. tomentella Hayata is unique in that it has four cytotypes (2n = 38, 40, 78, and 80) and a wide range of geographical distributions. The objective of this study was to uncover the genomic diversity among accessions of aneudiploid (2n = 38) and diploid (2n = 40) G. tomentella based on crossability rate, hybrid seed and seedling viability, meiotic chromosome pairing of F1 hybrids, and seed protein and protease inhibitor profiles. Aneudiploid and diploid G. tomentella accessions were divided into two (D1 and D2) and three [D3(A,B,C), D4, and D5] groups, respectively, based on previous isozyme studies. Crossability rate, intergenomic hybrid viability, degree of chromosome pairing, total seed protein profiles, and trypsin and chymotrypsin inhibitor banding patterns confirmed the isozyme grouping with minor disagreements. A consistent variation was not observed among the aneudiploid accessions in any method of analysis used in this study. Similarly, cytogenetic analysis and the total seed protein profiles did not show dissimilarity among the accessions from Papua New Guinea (PNG; the D3 group) and north of Mitchell River in Northern Queensland [N.Qld(n)]. However, trypsin and chymotrypsin inhibitor analysis revealed that the PNG accessions were distinctly different from N.Qld(n) accessions. The D4 and D5 group accessions were clearly distinguishable by both cytogenetic and biochemical methods. Thus, this study indicates the presence of four genomic groups among G. tomentella (2n = 38, 40) accessions, including the aneudiploids D1 and D2 in one group and diploids in three groups (D3, D4, and D5). These findings will be useful in further genome analysis and add to our present understanding of the biosystematics of the genus Glycine.