National Science Foundation-sponsored workshop report. Draft plan for soybean genomics

Recent efforts to coordinate and define a research strategy for soybean (Glycine max) genomics began with the establishment of a Soybean Genetics Executive Committee, which will serve as a communication focal point between the soybean research community and granting agencies. Secondly, a workshop was held to define a strategy to incorporate existing tools into a framework for advancing soybean genomics research. This workshop identified and ranked research priorities essential to making more informed decisions as to how to proceed with large scale sequencing and other genomics efforts. Most critical among these was the need to finalize a physical map and to obtain a better understanding of genome microstructure. Addressing these research needs will require pilot work on new technologies to demonstrate an ability to discriminate between recently duplicated regions in the soybean genome and pilot projects to analyze an adequate amount of random genome sequence to identify and catalog common repeats. The development of additional markers, reverse genetics tools, and bioinformatics is also necessary. Successful implementation of these goals will require close coordination among various working groups.     

Camptothecin analogs with bulky, hydrophobic substituents at the 7-position via a Grignard reaction

By developing a new synthetic procedure for introduction of side chains onto the camptothecin ring system, we were able to achieve the preparation of a number of analogs bearing bulky, hydrophobic groups directly attached to the 7-position. These include 7-tert-butylcamptothecin, 7-benzylcamptothecin and the corresponding 10,11-methylenedioxycamptothecins. This method involves the reaction of an appropriate orthoaminobenzonitrile with various Grignard reagents to give the corresponding orthoaminoketones. Friedlander condensation of the latter with the key tricyclic ketone leads to 7-substituted camptothecin analogs. We report the activity of these compounds as topoisomerase I poisons and their ability to inhibit growth of selected tumor cell lines.     

Partial reconstitution of human interstrand cross-link repair in vitro: characterization of the roles of RPA and PCNA

The repair of DNA interstrand cross-links (ICLs) remains largely ill-defined in higher eukaryotic cells. Previously, we have developed assays that can be used to monitor the early stages of processing of ICLs in vitro. Here, we have used P11 phosphocellulose chromatography to fractionate HeLa nuclear extracts and have subsequently reconstituted these assays with the resulting fractions. RPA and PCNA were found in a single fraction, and were the only factors in this fraction required for the reconstitution of these assays. The roles of RPA and PCNA in the formation of incisions at ICLs and in the subsequent DNA synthesis step were assessed. RPA was found to be essential for both stages of ICL processing indicating that it is required for lesion recognition and/or for the subsequent endonucleolytic processing. PCNA is required for the DNA synthesis stage and although it is not critical for the incision stage of the reaction it does enhance this step presumably by a stimulation of lesion recognition by MutSbeta. These findings define novel roles for RPA and PCNA in the processing of ICLs in mammalian cells.     

Osmoregulation and fungicide resistance: the Neurospora crassa os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue

Neurospora crassa osmosensitive (os) mutants are sensitive to high osmolarity and therefore are unable to grow on medium containing 4% NaCl. We found that os-2 and os-5 mutants were resistant to the phenylpyrrole fungicides fludioxonil and fenpiclonil. To understand the relationship between osmoregulation and fungicide resistance, we cloned the os-2 gene by using sib selection. os-2 encodes a putative mitogen-activated protein (MAP) kinase homologous to HOG1 and can complement the osmosensitive phenotype of a Saccharomyces cerevisiae hog1 mutant. We sequenced three os-2 alleles and found that all of them were null with either frameshift or nonsense point mutations. An os-2 gene replacement mutant also was generated and was sensitive to high osmolarity and resistant to phenylpyrrole fungicides. Conversely, os-2 mutants transformed with the wild-type os-2 gene could grow on media containing 4% NaCl and were sensitive to phenylpyrrole fungicides. Fludioxonil stimulated intracellular glycerol accumulation in wild-type strains but not in os-2 mutants. Fludioxonil also caused wild-type conidia and hyphal cells to swell and burst. These results suggest that the hyperosmotic stress response pathway of N. crassa is the target of phenylpyrrole fungicides and that fungicidal effects may result from a hyperactive os-2 MAP kinase pathway.     

Organization,expression and evolution of a disease resistance gene cluster in soybean

PCR amplification was previously used to identify a cluster of resistance gene analogues (RGAs) on soybean linkage group J. Resistance to powdery mildew (Rmd-c), Phytophthora stem and root rot (Rps2), and an ineffective nodulation gene (Rj2) map within this cluster. BAC fingerprinting and RGA-specific primers were used to develop a contig of BAC clones spanning this region in cultivar "Williams 82" [rps2, Rmd (adult onset), rj2]. Two cDNAs with homology to the TIR/NBD/LRR family of R-genes have also been mapped to opposite ends of a BAC in the contig Gm_Isb001_091F11 (BAC 91F11). Sequence analyses of BAC 91F11 identified 16 different resistance-like gene (RLG) sequences with homology to the TIR/NBD/LRR family of disease resistance genes. Four of these RLGs represent two potentially novel classes of disease resistance genes: TIR/NBD domains fused inframe to a putative defense-related protein (NtPRp27-like) and TIR domains fused inframe to soybean calmodulin Ca(2+)-binding domains. RT-PCR analyses using gene-specific primers allowed us to monitor the expression of individual genes in different tissues and developmental stages. Three genes appeared to be constitutively expressed, while three were differentially expressed. Analyses of the R-genes within this BAC suggest that R-gene evolution in soybean is a complex and dynamic process.     

Cloning and characterization of a testis and brain-specific isoform of mouse 3'-phosphoinositide-dependent protein kinase-1, mPDK-1 beta

3'-Phosphoinositide-dependent protein kinase-1 (PDK-1) phosphorylates and activates members of the protein kinase AGC family and plays a key role in receptor tyrosine kinase signaling. Here we report the cloning and characterization of a splice variant of mouse PDK-1, mPDK-1 beta. The cDNA encoding mPDK-1 beta contains two alternative start codons and translation from these start codons generates proteins that are, respectively, 27 or 51 amino acid residues shorter at the amino-terminus than the previously identified PDK-1 isolated from mouse liver (now renamed mPDK-1 alpha) [J. Biol. Chem. 274 (1999) 8117]. Analysis of mouse tissues shows that mPDK-1 beta is highly expressed in the testis and various functional regions of the brain. Expression of this isoform is increased in the brain of aged mice. Both mPDK-1 alpha and mPDK-1 beta are autophosphorylated at both serine and threonine residues in vitro and showed similar levels of tyrosine phosphorylation when co-expressed with either constitutively active Src or Fyn tyrosine kinases in cells. However, the mPDK-1 isoforms showed significant differences in their response to pervanadate- or insulin plus vanadate-stimulated tyrosine phosphorylation. Taken together, our findings suggest that the two PDK-1 isoforms may be differentially regulated in cells. The specific expression of mPDK-1 beta in mouse testis and brains of aged mice also suggests potential involvement of this kinase in regulating animal spermatogenesis and aging.     

Radiotherapy: effects on expanded skin

Although the combination of radiation and tissue expansion has been associated with a significant rate of complications, the specific pathophysiology has yet to be clearly elucidated. The objective of this study was to develop a model to identify and examine specific histologic changes associated with tissue expansion and irradiation. Rectangular 50-cc silicone tissue expanders were placed subcutaneously over the midline dorsum of 18 adult New Zealand white rabbits. Preoperative radiographic dosimetry demonstrated that the radiation portal was away from vital intraabdominal structures. The expanders were inflated with 10 cc of saline every other day for a total of 80 cc. Expanders were left in place for 2 to 3 weeks to allow fibrovascular capsule formation. The rabbits were then divided into three groups (six rabbits per group), each receiving one of three nonfractionated doses of radiation (20, 25, or 35 Gy). Half of the expanded skin was irradiated using a single dose, and the other half served as a nonirradiated control. Capsules and skin were harvested 6 weeks after the delivery of radiation, allowing the beginning of chronic radiation changes to occur. Using hematoxylin and eosin staining, histomorphometric analysis was performed. The data were analyzed using Student's test. Although irradiation did not affect dermal thickness, it did cause a statistically significant increase in epidermal thickness. At 20, 25, and 35 Gy the increase in epidermal thickness was 43, 90, and 130 percent, respectively. Although significant epidermal changes could be identified, capsular and dermal alterations were not evident. Further studies evaluating the long-term effects of alterations in capsular formation caused by radiation may be required.     

Frontally mediated control processes contribute to source memory retrieval

Remembering is a cognitively demanding task that requires the strategic selection of information from memory. In this issue of Neuron, Dobbins et al. present functional MRI (fMRI) data that shed insight into the specific, dissociated contributions of frontal regions to remembering.