A chromosomal genetic map of Rhizobium sp. NGR234 generated with Tn5-Mob

Summary Rhizobium sp. NGR234 in a fast-growing Rhizobium strain with a broad host range. The location and role of chromosomal genes involved in cellular metabolism or in the legume symbioses is unknown. We isolated a series of auxotrophic and antibiotic resistant mutants of NGR234 and utilized a chromosome mobilization system based on Tn5-Mob and pJB3JI; Tn5-Mob donor strains behaved like Hfr strains, transferring the chromosome polarly at high frequency from a fixed point of insertion. The use of four different strains with Tn5-Mob located at different nutritional loci in crosses with double auxotrophic recipients, allowed us to build up a circular linkage map of NGR234 based on relative recombination frequencies. Also, symbiotically important genes identified by site-directed mutagenesis, such as hemA and ntrA, could be located and mapped on the chromosome.Abbreviations Tc tetracycline - Sp spectinomycin - Rif rifampicin - Km kanamycin     

Using miniaturized radiotelemetry to discover the breeding grounds of the endangered New Zealand Storm Petrel Fregetta maoriana

Identification of breeding sites remains a critical step in species conservation, particularly in procellariiform seabirds whose threat status is of global concern. We designed and conducted an integrative radiotelemetry approach to uncover the breeding grounds of the critically endangered New Zealand Storm Petrel Fregetta maoriana (NZSP), a species considered extinct before its rediscovery in 2003. Solar‐powered automated radio receivers and hand‐held telemetry were used to detect the presence of birds on three island groups in the Hauraki Gulf near Auckland, New Zealand. At least 11 NZSP captured and radiotagged at sea were detected at night near Te Hauturu‐o‐Toi/Little Barrier Island with the detection of an incubating bird leading to the discovery of the first known breeding site for this species. In total, four NZSP breeding burrows were detected under mature forest canopy and three adult NZSP and two NZSP chicks were ringed. Telemetry data indicated NZSP showed strong moonlight avoidance behaviour over the breeding site, had incubation shifts of approximately 5 days and had a breeding season extending from February to June/July, a different season from other Procellariiformes in the region. Radiotelemetry, in combination with rigorously collected field data on species distribution, offers a valuable technique for locating breeding grounds of procellariiform seabirds and gaining insights into breeding biology while minimizing disturbance to sensitive species or damage to fragile habitat. Our study suggests an avenue for other breeding ground searches in one of the most threatened avian Orders, and highlights the general need for information on the location of breeding sites and understanding the breeding biology in data‐deficient birds.     

Isolation of single taste cells from lingual epithelium

A method is described for obtaining large numbers of isolatedtaste cells with identified polarity from lingual epithelium.The procedure involves incubating lingual epithelium in collagenase,staining the apical surface with fluorescein-conjugated wheatgerm agglutinin (FTTC-WGA), peeling non-gustatory surface epitheliumfrom the underlying taste buds and connective tissue, and dissociatingisolated taste buds with Ca²⁺-free saline. Isolated taste cellsretain their characteristic morphology for at least 30 min afterdissociation, and the apical specialization can be identifiedas a single patch of fluorescence usually located at the tipof an elongate process. Isolated taste cells are amenable tostudy with the patch-clamp technique, and whole-cell patch-clamprecordings show that isolated taste cells have membrane propertiessimilar to taste cells of intact lingual epithelium. Evidenceis presented that FITC-WGA staining does not alter the voltage-dependentionic currents of the taste cell membrane.     

RAC1 induces nuclear alterations through the LINC complex to enhance melanoma invasiveness

RHO GTPases are key regulators of the cytoskeletal architecture, which impact a broad range of biological processes in malignant cells including motility, invasion, and metastasis, thereby affecting tumor progression. One of the constraints during cell migration is the diameter of the pores through which cells pass. In this respect, the size and shape of the nucleus pose a major limitation. Therefore, enhanced nuclear plasticity can promote cell migration. Nuclear morphology is determined in part through the cytoskeleton, which connects to the nucleoskeleton through the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Here, we unravel the role of RAC1 as an orchestrator of nuclear morphology in melanoma cells. We demonstrate that activated RAC1 promotes nuclear alterations through its effector PAK1 and the tubulin cytoskeleton, thereby enhancing migration and intravasation of melanoma cells. Disruption of the LINC complex prevented RAC1-induced nuclear alterations and the invasive properties of melanoma cells. Thus, RAC1 induces nuclear morphology alterations through microtubules and the LINC complex to promote an invasive phenotype in melanoma cells.     

Annexin A2-S100A10 heterotetramer, a novel substrate of thioredoxin

The binding of plasminogen activators and plasminogen to the cell surface results in the rapid generation of the serine protease plasmin. Plasmin is further degraded by an autoproteolytic reaction, resulting in the release of an angiostatin, A61 (Lys78-Lys468). Previously, we demonstrated that the annexin A2-S100A10 heterotetramer (AIIt) stimulates the release of A61 from plasmin by promoting the autoproteolytic cleavage of the Lys468-Gly469 bond and reduction of the plasmin Cys462-Cys541 disulfide (Kwon, M., Caplan, J. F., Filipenko, N. R., Choi, K. S., Fitzpatrick, S. L., Zhang, L., and Waisman, D. M. (2002) J. Biol. Chem. 277, 10903-10911). Mechanistically, it was unclear if AIIt promoted a conformational change in plasmin, resulting in contortion of the plasmin disulfide, or directly reduced the plasmin disulfide. In the present study, we show that AIIt thiols are oxidized during the reduction of plasmin disulfides, establishing that AIIt directly participates in the reduction reaction. Incubation of HT1080 cells with plasminogen resulted in the rapid loss of thiol-specific labeling of AIIt by 3-(N-maleimidopropionyl)biocytin. The plasminogen-dependent oxidation of AIIt could be attenuated by thioredoxin. Thioredoxin reductase catalyzed the transfer of electrons from NADPH to the oxidized thioredoxin, thus completing the flow of electrons from NADPH to AIIt. Therefore, we identify AIIt as a substrate of the thioredoxin system and propose a new model for the role of AIIt in the redox-dependent processing of plasminogen and generation of an angiostatin at the cell surface.     

Interspecific pollen competition as a reproductive barrier between sympatric species of Helianthus (Asteraceae)

Artificial crosses between Helianthus annuus and H. petiolaris using 1:9, 1:1, and 9:1 mixtures of intraspecific: interspecific pollen were conducted to determine the role of interspecific pollen competition as a reproductive barrier in Helianthus. Of 1,245 achenes analyzed from the pollen competition experiments, only 49 were hybrids. The number of hybrids observed was significantly less than expectations for all three pollen mixtures, regardless of the identity of maternal parent (P < 0.01). Stigma age and pollen ratio had no significant impact on hybrid frequency. However, hybrids were significantly more frequent with H. annuus than with H. petiolaris as the maternal parent (P < 0.01). Analysis of pollen tube growth rates revealed no differences in the rate of growth of intraspecific vs. interspecific pollen. Likewise, pollinations with either intraspecific or interspecific pollen or with different pollen ratios did not affect the percentage of filled achenes. Thus, the mechanism responsible for selective fertilization by intraspecific pollen in mixed pollen loads remains unclear. Nonetheless, these findings suggest that interspecific pollen competition plays an important role in controlling the formation of hybrids between H. annuus and H. petiolaris and may partially account for patterns or differential cytoplasmic vs. nuclear introgression in Helianthus.     

The Wilderness Act and Fish Stocking: An Overview of Legislation, Judicial Interpretation, and Agency Implementation

Many high-elevation lakes in designated wilderness are stocked with native and nonnative fish by state fish and game agencies to provide recreational fishing opportunities. In several areas, this practice has become controversial with state wildlife managers who support historical recreational use of wilderness, federal wilderness managers who assert that stocking compromises some of the ecological and social values of wilderness, and different public groups that support one or the other position. Herein we examine this controversy from the perspective of the 1964 Wilderness Act, its judicial interpretation, the policies of the federal agencies, and formal agreements between federal and state agencies. Although some state stocking programs restore native fish populations, other programs may compromise some of the ecological and social values of wilderness areas. Further, although current federal regulations recognize state authority for fish stocking, judicial interpretation gives federal agencies the authority for direct involvement in decisions regarding fish stocking in wilderness. Where there are differences of opinion between state and federal managers, this judicial interpretation strongly points to the need for improved cooperation, communication, and coordination between state wildlife managers and federal wilderness managers to balance recreational fishing opportunities and other wildlife management activities with wilderness values. Received 28 March 2000; Accepted 16 August 2000.     

First Sequenced Mitochondrial Genome from the Phylum Acanthocephala(Leptorhynchoides thecatus) and Its Phylogenetic Position Within Metazoa

The complete sequence of the mitochondrial genome of Leptorhynchoides thecatus (Acanthocephala) was determined, and a phylogenetic analysis was carried out to determine its placement within Metazoa. The genome is circular, 13,888 bp, and contains at least 36 of the 37 genes typically found in animal mitochondrial genomes. The genes for the large and small ribosomal RNA subunits are shorter than those of most metazoans, and the structures of most of the tRNA genes are atypical. There are two significant noncoding regions (377 and 294 bp), which are the best candidates for a control region; however, these regions do not appear similar to any of the control regions of other animals studied to date. The amino acid and nucleotide sequences of the protein coding genes of L. thecatus and 25 other metazoan taxa were used in both maximum likelihood and maximum parsimony phylogenetic analyses. Results indicate that among taxa with available mitochondrial genome sequences, Platyhelminthes is the closest relative to L. thecatus, which together are the sister taxon of Nematoda; however, long branches and/or base composition bias could be responsible for this result. The monophyly of Ecdysozoa, molting organisms, was not supported by any of the analyses. This study represents the first mitochondrial genome of an acanthocephalan to be sequenced and will allow further studies of systematics, population genetics, and genome evolution.Reviewing Editor: Dr. Rafael Zardoya The entire genome sequence has been deposited with the GenBank Data Libraries under-accession number AY562383.