Genome organization in dicots. II. Arabidopsis as a ’bridging species’ to resolve genome evolution events among legumes

Analysis of molecular linkage groups within the soybean (Glycine max L. Merr.) genome reveals many homologous regions, reflecting the ancient polyploidy of soybean. The fragmented arrangement of the duplicated regions suggests that extensive rearrangements, as well as additional duplications, have occurred since the initial polyploidization event. In this study we used comparisons between homoeologous regions in soybean, and the homologous regions in the related diploids Phaseolus vulgaris and Vigna radiata, to elucidate the evolutionary history of the three legume genomes. Our results show that there is not only conservation of large regions of the genomes but that these conserved linkage blocks are also represented twice in the soybean genome. To gain a better understanding of the process of genome evolution in dicots, molecular comparisons have been extended to another well-studied species, Arabidopsis thaliana. Interestingly, the conserved regions we identified in the legume species are also relatively conserved in Arabidopsis. Our results suggest that there is conservation of blocks of DNA between species as distantly related as legumes and brassicas, representing 90 million years of divergence. We also present evidence for an additional, presumably earlier, genome duplication in soybean. These duplicated regions were only recognized by using Arabidopsis as a ’bridging’ species in the genome comparisons. Received: 10 October 2000 / Accepted: 13 January 2001     

Identification, structure, and differential expression of members of a BURP domain containing protein family in soybean.

Expressed sequence tags (ESTs) exhibiting homology to a BURP domain containing gene family were identified from the Glycine max (L.) Merr. EST database. These ESTs were assembled into 16 contigs of variable sizes and lengths. Consistent with the structure of known BURP domain containing proteins, the translation products exhibit a modular structure consisting of a C-terminal BURP domain, an N-terminal signal sequence, and a variable internal region. The soybean family members exhibit 35-98% similarity in a -100-amino-acid C-terminal region, and a phylogenetic tree constructed using this region shows that some soybean family members group together in closely related pairs, triplets, and quartets, whereas others remain as singletons. The structure of these groups suggests that multiple gene duplication events occurred during the evolutionary history of this family. The depth and diversity of G. max EST libraries allowed tissue-specific expression patterns of the putative soybean BURPs to be examined. Consistent with known BURP proteins, the newly identified soybean BURPs have diverse expression patterns. Furthermore, putative paralogs can have both spatially and quantitatively distinct expression patterns. We discuss the functional and evolutionary implications of these findings, as well as the utility of EST-based analyses for identifying and characterizing gene families.     

A compilation of soybean ESTs: generation and analysis.

Whole-genome sequencing is fundamental to understanding the genetic composition of an organism. Given the size and complexity of the soybean genome, an alternative approach is targeted random-gene sequencing, which provides an immediate and productive method of gene discovery. In this study, more than 120000 soybean expressed sequence tags (ESTs) generated from more than 50 cDNA libraries were evaluated. These ESTs coalesced into 16928 contigs and 17336 singletons. On average, each contig was composed of 6 ESTs and spanned 788 bases. The average sequence length submitted to dbEST was 414 bases. Using only those libraries generating more than 800 ESTs each and only those contigs with 10 or more ESTs each, correlated patterns of gene expression among libraries and genes were discerned. Two-dimensional qualitative representations of contig and library similarities were generated based on expression profiles. Genes with similar expression patterns and, potentially, similar functions were identified. These studies provide a rich source of publicly available gene sequences as well as valuable insight into the structure, function, and evolution of a model crop legume genome.     

Soybean genomic survey: BAC-end sequences near RFLP and SSR markers.

We are building a framework physical infrastructure across the soybean genome by using SSR (simple sequence repeat) and RFLP (restriction fragment length polymorphism) markers to identify BACs (bacterial artificial chromosomes) from two soybean BAC libraries. The libraries were prepared from two genotypes, each digested with a different restriction enzyme. The BACs identified by each marker were grouped into contigs. We have obtained BAC- end sequence from BACs within each contig. The sequences were analyzed by the University of Minnesota Center for Computational Genomics and Bioinformatics using BLAST algorithms to search nucleotide and protein databases. The SSR-identified BACs had a higher percentage of significant BLAST hits than did the RFLP-identified BACs. This difference was due to a higher percentage of hits to repetitive-type sequences for the SSR-identified BACs that was offset in part, however, by a somewhat larger proportion of RFLP-identified significant hits with similarity to experimentally defined genes and soybean ESTs (expressed sequence tags). These genes represented a wide range of metabolic functions. In these analyses, only repetitive sequences from SSR-identified contigs appeared to be clustered. The BAC-end sequences also allowed us to identify microsynteny between soybean and the model plants Arabidopsis thaliana and Medicago truncatula. This map-based approach to genome sampling provides a means of assaying soybean genome structure and organization.     

Induction of B-A transitions of deoxyoligonucleotides by multivalent cations in dilute aqueous solution.

Circular dichroism (CD) spectra of d(CCCCGGGG) in the presence of Co(NH3)6(3+) are very similar to spectra of r(CCCCGGGG). In contrast, B-form characteristics are observed for d(CCCCGGGG) in the presence of Na+ and Mg2+, even at high salt concentrations. Spermidine induces modest changes of the CD of d(CCCCGGGG). The NMR chemical shifts of the nonexchangeable protons of d(CCCCGGGG) in the absence and presence of Co(NH3)6(3+) were assigned by proton two-dimensional (2D) NOESY and COSY measurements. The chemical shifts of the GH8 protons of d(CCCCGGGG) move upfield upon titration with Co(NH3)6Cl3. The sums of the sugar H1' coupling constants decrease with added Co(NH3)6Cl3. Cross peak intensities in the 2D proton NOESY spectra show a transformation from B-DNA to A-DNA characteristics upon the addition of Co(NH3)6Cl3. The temperature-dependent 59Co transverse and longitudinal relaxation rates demonstrate that Co(NH3)6(3+) is site-bound to the oligomer. Such localization is not a general feature of Co(NH3)6(3+) binding to oligonucleotides. 59Co NMR relaxation and CD measurements demonstrate chiral discrimination by d(CCCCGGGG) for the two stereoisomers of Co(en)3(3+). Both stereoisomers bind tightly as judged by 59Co NMR, and both cause large (but nonequivalent) changes in the CD of this oligomer.     

Genome conservation among three legume genera detected with DNA markers.

A set of 219 DNA clones derived from mungbean (Vigna radiata), cowpea (V. unguiculata), common bean (Phaseolus vulgaris), and soybean (Glycine max) were used to generate comparative linkage maps among mungbean, common bean, and soybean. The maps allowed an assessment of linkage conservation and collinearity among the three genomes. Mungbean and common bean, both of the subtribe Phaseolinae, exhibited a high degree of linkage conservation and preservation of marker order. Most linkage groups of mungbean consisted of only one or two linkage blocks from common bean (and vice versa). The situation was significantly different with soybean, a member of the subtribe Glycininae. Mungbean and common bean linkage groups were generally mosaics of short soybean linkage blocks, each only a few centimorgans in length. These results suggest that it would be fruitful to join maps of mungbean and common bean, while knowledge of conserved genomic blocks would be useful in increasing marker density in specific genomic regions for all three genera. These comparative maps may also contribute to enhanced understanding of legume evolution.     

Structural characterization of inhibitor complexes with checkpoint kinase 2 (Chk2), a drug target for cancer therapy

Chk2 (checkpoint kinase 2) is a serine/threonine kinase that participates in a series of signaling networks responsible for maintaining genomic integrity and responding to DNA damage. The development of selective Chk2 inhibitors has recently attracted much interest as a means of sensitizing cancer cells to current DNA-damaging agents used in the treatment of cancer. Additionally, selective Chk2 inhibitors may reduce p53-mediated apoptosis in normal tissues, thereby helping to mitigate adverse side effects from chemotherapy and radiation. Thus far, relatively few selective inhibitors of Chk2 have been described and none have yet progressed into clinical trials. Here, we report crystal structures of the catalytic domain of Chk2 in complex with a novel series of potent and selective small molecule inhibitors. These compounds exhibit nanomolar potencies and are selective for Chk2 over Chk1. The structures reported here elucidate the binding modes of these inhibitors to Chk2 and provide information that can be exploited for the structure-assisted design of novel chemotherapeutics.     

Identification of the guanine binding domain peptide of the GTP-binding site of glucagon.

Glucagon, a peptide hormone synthesized and secreted by alpha islet cells, regulates glucose homeostasis by several mechanisms. Using [gamma 32P]8N3GTP, a proven photoaffinity probe for GTP, a specific nucleotide binding site on human glucagon was detected that showed preference for GTP. Half-maximal saturation of photoinsertion into the polypeptide hormone was at 8-12 microM with either [alpha 32P]8N3GTP or [gamma 32P]8N3GTP. GTP protected photolabeling with an apparent kd of 15 microM, whereas ATP was less effective as a protector, exhibiting an apparent kd of about 30 microM. Maximal protection by GTP and ATP was over 90%. UTP, CTP, GDP, ADP, GMP, AMP, guanosine, adenosine, guanine, and adenine were much less effective protectors, indicating that binding is specific for purine nucleoside triphosphates, particularly GTP. Mg2+ at 150 microM enhanced photoinsertion (twofold), whereas at 2-10 mM, it inhibited photoinsertion. Both Ca2+ and Zn2+ at 0.2 mM decreased photoinsertion about 45%. Purification of chymotryptic and tryptic digests of photolabeled glucagon by reverse-phase high performance liquid chromatography (HPLC) revealed that the N-terminal peptide, HSQGTF, was the only peptide region covalently photomodified by [32P]8N3GTP. GTP, if present during photolysis, greatly reduced both photoinsertion into glucagon and the amount of radiolabeled peptide recovered on HPLC. The specificity of binding to the N-terminal region is suggestive of a physiological role for a glucagon-GTP complex in the mechanism of action of this hormone.     

Insertion and excision of Bacteroides conjugative chromosomal elements.

Many strains of Bacteroides harbor large chromosomal elements that can transfer themselves from the chromosome of the donor to the chromosome of the recipient. Most of them carry a tetracycline resistance (Tcr) gene and have thus been designated Tcr elements. In the present study, we have used transverse alternating field electrophoresis to show that all but one of the Tcr elements screened were approximately 70 to 80 kbp in size. The exception (Tcr Emr 12256) was 150 to 200 kbp in size and may be a hybrid element. All of the Tcr elements inserted in more than one site, but insertion was not random. The Tcr elements sometimes cotransfer unlinked chromosomal segments, or nonreplicating Bacteroides units (NBUs). Transverse alternating field electrophoresis analysis showed that insertion of NBUs was not random and that the NBUs did not insert near the Tcr element. Although attempts to clone one or both ends of a Tcr element have not been successful, ends of a cryptic element (XBU4422) were cloned previously and shown to be homologous to the ends of Tcr elements. We have obtained DNA sequences of junction regions between XBU4422 and its target from several different insertions. Comparison of junction sequences with target sequences showed that no target site duplication occurred during insertion and that XBU4422 carried 4 to 5 bp of adjacent chromosomal DNA when it excised from the chromosome and inserted in a plasmid. We identified a short region of sequence similarity between one of the ends of XBU4422 and its target site that may be important for insertion. This sequence contained an 8-bp segment that was identical to the recombinational hot spot sequence on Tn21. XBU4422 could exise itself from plasmids into which it inserted. In most cases, the excision left a single additional A behind in the target site, but precise excision was seen in one case.