Indigenous peoples and local communities as partners in the sequencing of global eukaryotic biodiversity.

The aim to sequence, catalog, and characterize the genomes of all of Earth's eukaryotic biodiversity is the shared mission of many ongoing large-scale biodiversity genomics initiatives. Reference genomes of global flora and fauna have the potential to inform a broad range of major issues facing both biodiversity and humanity, such as the impact of climate change, the conservation of endangered species and ecosystems, public health crises, and the preservation and enhancement of ecosystem services. Biodiversity is dramatically declining: 28% of species being assessed by the IUCN are threatened with extinction, and recent reports suggest that a transformative change is needed to conserve and protect what remains. To provide a collective and global genomic response to the biodiversity crisis, many biodiversity genomics initiatives have come together, creating a network of networks under the Earth BioGenome Project. This network seeks to expedite the creation of an openly available, "public good" encyclopedia of high-quality eukaryotic reference genomes, in the hope that by advancing our basic understanding of nature, it can lead to the transformational scientific developments needed to conserve and protect global biodiversity. Key to completing this ambitious encyclopedia of reference genomes, is the ability to responsibly, ethically, legally, and equitably access and use samples from all of the eukaryotic species across the planet, including those that are under the custodianship of Indigenous Peoples and Local Communities. Here, the biodiversity genomics community is subject to the provisions codified in international, national, and local legislations and customary community norms, principles, and protocols. We propose a framework to support biodiversity genomic researchers, projects, and initiatives in building trustworthy and sustainable partnerships with communities, providing minimum recommendations on how to access, utilize, preserve, handle, share, analyze, and communicate samples, genomics data, and associated Traditional Knowledge obtained from, and in partnership with, Indigenous Peoples and Local Communities across the data-lifecycle.

First Report of Infection of Poison Hemlock and Celery by Apium virus Y in Washington State.

A commercial field of celery (Apium graveolens var. dulce) cvs. Conquistador and Sabroso was planted with sets between 1 June and 10 July 2004 in Pierce County in western Washington (WA). In late August, many plants were stunted and showed chlorotic line patterns. One symptomatic plant and five nonsymptomatic plants were transferred to a greenhouse and grown at 22°C with supplemental lighting to extend day length to 16 h; foliage was trimmed back. The symptomatic plant and three nonsymptomatic plants developed a distinctive chlorotic line pattern when new foliage emerged in February. Two plants remained nonsymptomatic. Young foliage was tested by ELISA with the general potyvirus monoclonal antibody (Agdia, Inc., Elkhart, IN). All symptomatic plants yielded a positive result and the two nonsymptomatic plants were negative. Celery mosaic virus (CeMV) was previously reported to be widespread in WA (3), but primers specific for CeMV did not yield amplicons in reverse transcription (RT)-PCR from RNA isolated from symptomatic leaf tissue (RNeasy Plant Mini Kit: QIAGEN, Valencia, CA). General potyvirus primers (1) were used to amplify ≈1,700 nucleotides from the 3' terminus of the virus genome adjacent to the poly-A tail. Six amplicons from each of three independent reactions were cloned into pCR2.1 (Invitrogen, Carlsbad, CA) and sequenced (GenBank Accession No. FJ010827). Comparison with the nucleotide sequence database revealed 98 and 97% identity to Australian isolates of Apium virus Y (ApVY) (family Potyviridae) from parsley (GenBank Accession No. AF207594) and poison hemlock (Conium maculatum) (GenBank Accession No. AY049716) (4) and 91% identity to North American isolates of ApVY from celery and Ammi majus reported from California (GenBank Accession No. EU515126) and Florida (GenBank Accession No. EU255632), respectively. To our knowledge, this is the first report of a natural infection of celery by ApVY in WA. No other potyvirus sequences were identified in RT-PCR products from symptomatic celery. In 2008, in an effort to locate local samples of CeMV, poison hemlock plants were randomly collected in Benton, Walla Walla, and Whitman counties of eastern WA. No symptoms were observed and no CeMV was detected by RT-PCR in any of these plants. No ApVY was detected in 10 of 10 poison hemlock collected from Walla Walla County, but based on sequence analysis of RT-PCR amplicons, two of two plants collected from Benton and Whitman counties were infected with ApVY. In contrast to the WA isolates from celery, sequences from these poison hemlock plants (GenBank Accession No. FJ010828) were 98% identical to previously reported North American isolates of ApVY (GenBank Accession Nos. EU515126 and EU255632) and only 91% identical to Australian isolates (GenBank Accession Nos. AF207594 and AY049716). To our knowledge, this is the first report of ApVY in WA and the first report of a natural infection of poison hemlock in the United States. The celery and poison hemlock isolates reported in this study were from different geographic regions of the state and were only 91% identical. As is the case for other potyviruses (2), weeds such as poison hemlock may serve as a reservoir of ApVY in eastern WA where many plants of the family Apiaceae are grown commercially. References: (1) J. Chen et al. Arch. Virol. 146:757, 2001. (2) W. E. Howell and G. I. Mink. Plant Dis. Rep. 61:217, 1977. (3) W. E. Howell and G. I. Mink. Plant Dis. 65:277, 1981. (4) J. Moran et al. Arch. Virol. 147:1855, 2002.

Assessment of Barriers to Prevent the Development of Potato Tuber Blight Caused by Phytophthora infestans.

Experiments were conducted in an irrigated, sandy loam soil to evaluate mulches and hill sizes as barriers to prevent the development of potato tuber blight caused by Phytophthora infestans. In mulching experiments, five treatments were applied to field plots of cv. Red LaSoda: 1, no mulch; 2, polyurethane spray foam in an 8-cm-diameter area immediately surrounding the plant stem; 3, black polyethylene film over the entire hill except near the stem; 4, a combination of treatments 2 and 3; and 5, a water-permeable, agricultural textile treated with copper hydroxide applied over the same hill area as in treatment 3. In 1998, the incidence of tuber blight in plots mulched with black film (treatments 3 and 4) averaged 32% compared with 56% in plots without this mulch (treatments 1 and 2). In 1999, incidence of tuber blight in plots with and without black film averaged 9 and 20%, respectively. Mulching the stem area with spray foam (treatments 2 and 3) did not reduce the incidence of blighted tubers when compared with the appropriate control. The copper-treated textile mulch (treatment 5) provided reductions in the incidence of tuber blight similar to those observed with the use of black polyethylene film. In a hill size experiment conducted once in 1998 and twice in 1999, three hill size treatments were established on cvs. Red LaSoda, Shepody, and Russet Burbank. Red LaSoda was the most susceptible and Russet Burbank the least susceptible to tuber blight. Comparison of blight incidence in tubers classified by depth in the hill revealed few differences among the hill size treatments, although over all treatments, tubers covered with more than 15 cm of soil had a lower incidence of blight (1 to 14%) than tubers with less soil cover (13 to 59%). Most tuber infections were apparently initiated in eyes and were not concentrated on a portion of the tuber such as the stolon (proximal) or distal end. The fact that black film and textile mulches reduced tuber infection indicates that inoculum of P. infestans can move from foliage to tubers through soil and that inoculum movement is not limited to large channels in the hill such as those created by the potato stems. The mulch treatments, however, provided only partial protection of tubers, limiting the practicality of such treatments to commercial producers. Hill size treatments had little effect on tuber blight incidence, indicating that adequate suppression of tuber infection in an environment conducive to late blight may be inseparably linked to adequate suppression of the foliar phase.

Enhancement of cell interactions with collagen/glycosaminoglycan matrices by RGD derivatization.

The interaction of three cell types important to the wound repair process with collagen/glycosaminoglycan (GAG) dermal regeneration matrices covalently modified with an Arg-Gly-Asp (RGD)-containing peptide was characterized. Function-blocking monoclonal antibodies directed against various integrin subunits were used to demonstrate that human fibroblasts attached to the unmodified matrix through the integrin, alpha2beta1. Human endothelial cells and human keratinocytes, however, attached minimally to the unmodified matrix. After modification of the collagen/GAG matrix with RGD-containing peptide, endothelial cells and keratinocytes attached and spread well on the matrix. This attachment was RGD dependent as evidenced by essentially complete inhibition with competing soluble peptide. In terms of overall cell number, fibroblast cell attachment remained unchanged on the RGD peptide-modified matrix compared to the unmodified material. Antibody and peptide inhibition studies demonstrate, however, that attachment to the modified matrix was mediated by both alpha2beta1 and RGD-binding integrins. We have successfully introduced a specific RGD receptor-mediated attachment site on collagen/GAG dermal regeneration matrices, resulting in enhanced cell interaction of important wound healing cell types. This modification could have important implications for the performance of these matrices in promoting dermal regeneration.

Characterization of a hyaluronic acid-Arg-Gly-Asp peptide cell attachment matrix.

We have developed a method to modify cross-linked hyaluronic acid with peptides containing the Arg-Gly-Asp sequence. The material created by this process is a three-dimensional porous matrix capable of supporting integrin receptor-mediated cell attachment. Peptide density can be controlled by varying the reaction conditions during peptide immobilization. Following cell attachment, cells actively proliferate and colonize the pores of the matrix. This material should prove useful for the maintenance of cells on a chemically defined three-dimensional substrate or as a scaffold for enhancing tissue repair.

The alpha-helical rod domain of human lamins A and C contains a chromatin binding site.

We examined regions of human lamins A and C involved in binding to surfaces of mitotic chromosomes. An Escherichia coli expression system was used to produce full-length lamin A and lamin C, and truncated lamins retaining the central alpha-helical rod domain (residues 34-388) but lacking various amounts of the amino-terminal 'head' and carboxy-terminal 'tail' domains. We found that lamin A, lamin C and lamin fragments lacking the head domain and tail sequences distal to residue 431 efficiently assembled into paracrystals and strongly associated with mitotic chromosomes. Furthermore, the lamin rod domain also associated with chromosomes, although efficient chromosome coating required the pH 5-6 conditions needed to assemble the rod into higher order structures. Biochemical assays showed that chromosomes substantially reduced the critical concentration for assembly of lamin polypeptides into pelletable structures. Association of the lamin rod with chromosomes was abolished by pretrypsinization of chromosomes, and was not seen for vimentin (which possesses a similar rod domain). These data demonstrate that the alpha-helical rod of lamins A and C contains a specific chromosome binding site. Hence, the central rod domain of intermediate filament proteins can be involved in interactions with other cellular structures as well as in filament assembly.

An endoplasmic reticulum-specific cyclophilin.

Cyclophilin is a ubiquitously expressed cytosolic peptidyl-prolyl cis-trans isomerase that is inhibited by the immunosuppressive drug cyclosporin A. A degenerate oligonucleotide based on a conserved cyclophilin sequence was used to isolate cDNA clones representing a ubiquitously expressed mRNA from mice and humans. This mRNA encodes a novel 20-kDa protein, CPH2, that shares 64% sequence identity with cyclophilin. Bacterially expressed CPH2 binds cyclosporin A and is a cyclosporin A-inhibitable peptidyl-prolyl cis-trans isomerase. Cell fractionation of rat liver followed by Western blot (immunoblot) analysis indicated that CPH2 is not cytosolic but rather is located exclusively in the endoplasmic reticulum. These results suggest that cyclosporin A mediates its effect on cells through more than one cyclophilin and that cyclosporin A-induced misfolding of T-cell membrane proteins normally mediated by CPH2 plays a role in immunosuppression.

Lamins A and C bind and assemble at the surface of mitotic chromosomes.

To study a possible interaction of nuclear lamins with chromatin, we examined assembly of lamins A and C at mitotic chromosome surfaces in vitro. When a postmicrosomal supernatant of metaphase CHO cells containing disassembled lamins A and C is incubated with chromosomes isolated from mitotic Chinese hamster ovary cells, lamins A and C undergo dephosphorylation and uniformly coat the chromosome surfaces. Furthermore, when purified rat liver lamins A and C are dialyzed with mitotic chromosomes into a buffer of physiological ionic strength and pH, lamins A and C coat chromosomes in a similar fashion. In both cases a lamin-containing supramolecular structure is formed that remains intact when the chromatin is removed by digestion with micrococcal nuclease and extraction with 0.5 M KCl. Lamins associate with chromosomes at concentrations approximately eightfold lower than the critical concentration at which they self-assemble into insoluble structures in the absence of chromosomes, indicating that chromosome surfaces contain binding sites that promote lamin assembly. These binding sites are destroyed by brief treatment of chromosomes with trypsin or micrococcal nuclease. Together, these data suggest the existence of a specific lamin-chromatin interaction in cells that may be important for nuclear envelope reassembly and interphase chromosome structure.

Alteration of cellular adhesion by heat shock.

Chinese hamster ovary (CHO) cells were analyzed for their ability to reassemble microfilament bundles, to remain attached to a tissue culture surface, or to initiate and complete attachment onto a substrate after heat shock (45 degrees C/10 min). The cells remained attached to the tissue culture surface during and after the heat shock while the actin microfilament bundles were reversibly disrupted. Heat shock inhibited the ability of the cells to initiate and complete attachment onto a new tissue culture surface or onto a plastic surface coated with vitronectin. An inspection of the proteins present in substrate-attached material (SAM) revealed 11 major proteins containing glucosamine whose apparent Mr values were 250,000, 200,000, 150,000, 140,000, 90,000, 86,000, 82,000, 68,000, 54,000, 47,000, and 46,000. Three of the proteins (p200, p150, and p46) bound to wheat germ agglutinin while p150 and p140 bound to concanavalin A. The composition of the 11 proteins of the SAM fraction synthesized previous to the heat shock was not altered during heat shock. However, the appearance of the newly synthesized proteins in the SAM fraction was delayed by heat shock (0.5 h for p150 and 6 h for p82). The ability of heat-shocked cells to reattach onto a vitronectin-coated surface correlated with the appearance of newly synthesized p150 and p82 in the SAM fraction. Our results suggest that in addition to the microfilament bundles, heat shock may reversibly disrupt the cellular adhesion site. Further, p150 and p82, proteins whose appearance in the SAM fraction is delayed by heat shock, may be involved in the cellular attachment onto substrates, including vitronectin.

Ornithine decarboxylase production in vitro by using mouse cDNA.

Microgram quantities of ornithine decarboxylase (ODC, EC 4.1.1.17)-specific mRNA were synthesized by transcription techniques in vitro, by using a plasmid containing mouse cDNA coding for this enzyme. The homogeneous RNA preparation was then used for cell-free synthesis of ODC protein, in rabbit reticulocyte lysates. Analysis of products translated in vitro by polyacrylamide-gel electrophoresis revealed predominantly one protein produced, with Mr approx. 54,000, which was immunoprecipitable by anti-ODC serum. Two-dimensional gel-electrophoretic analysis showed that the protein ODC synthesized in vitro had a pI of approx. 5.4, similar to the native enzyme isolated from mouse tissues. In addition, quantification of activity and protein amount showed that the enzyme synthesized in vitro had a specific activity of approx. 63,000 units (nmol/min)/mg, consistent with the purified mouse kidney enzyme's specific activity of approx. 47,000 units/mg. An average of nearly 200 pg of ODC protein was produced in vitro from various RNA preparations. These data demonstrate that ODC-specific mRNA and active ODC protein can be produced by 'in vitro' technology, which should prove useful in studying functional and structural characteristics of these molecules.

Spermidine mediates degradation of ornithine decarboxylase by a non-lysosomal, ubiquitin-independent mechanism.

The mechanism of spermidine-induced ornithine decarboxylase (ODC, E.C. 4.1.1.17) inactivation was investigated using Chinese hamster ovary (CHO) cells, maintained in serum-free medium, which display a stabilization of ODC owing to the lack of accumulation of putrescine and spermidine (Glass and Gerner: Biochem. J., 236:351-357, 1986; Sertich et al.: J. Cell Physiol., 127:114-120, 1986). Treatment of cells with 10 microM exogenous spermidine leads to rapid decay of ODC activity accompanied by a parallel decrease in enzyme protein. Analysis of the decay of [35S]methionine-labeled ODC and separation by two-dimensional electrophoresis revealed no detectable modification in ODC structure during enhanced degradation. Spermidine-mediated inactivation of ODC occurred in a temperature-dependent manner exhibiting pseudo-first-order kinetics over a temperature range of 22-37 degrees C. In cultures treated continuously, an initial lag was observed after treatment with spermidine, followed by a rapid decline in activity as an apparent critical concentration of intracellular spermidine was achieved. Treating cells at 22 degrees C for 3 hours with 10 microM spermidine, followed by removal of exogenous polyamine, and then shifting to varying temperatures, resulted in rates of ODC inactivation identical with that determined with a continuous treatment. Arrhenius analysis showed that polyamine mediated inactivation of ODC occurred with an activation energy of approximately 16 kcal/mol. Treatment of cells with lysosomotrophic agents (NH4Cl, chloroquine, antipain, leupeptin, chymostatin) had no effect on ODC degradation. ODC turnover was not dependent on ubiquitin-dependent proteolysis. Shift of ts85 cells, a temperature-sensitive mutant for ubiquitin conjugation, to 39 degrees C (nonpermissive for ubiquitin-dependent proteolysis) followed by addition of spermidine led to a rapid decline in ODC activity, with a rate similar to that seen at 32 degrees C (the permissive temperature). In contrast, spermidine-mediated ODC degradation was substantially decreased by inhibitors of protein synthesis (cycloheximide, emetine, and puromycin). These data support the hypothesis that spermidine regulates ODC degradation via a mechanism requiring new protein synthesis, and that this occurs via a non-lysosomal, ubiquitin-independent pathway.

Polyamine-mediated turnover of ornithine decarboxylase in Chinese-hamster ovary cells.

We have used Chinese-hamster ovary (CHO) cells maintained in a chemically defined medium to study the regulation of ornithine decarboxylase (ODC) by polyamines. Cells maintained in the defined medium had no detectable putrescine, and approx. 1-3 units of ODC activity/10(6) cells, where 1 unit corresponds to 1 nmol of substrate decarboxylated in 30 min. The defined medium is ornithine-deficient, thus limiting the exogenous substrate for ODC, and subsequently decreasing intracellular polyamine accumulation. Restoration of intracellular putrescine and increased formation of spermidine by addition of exogenous ornithine or putrescine led to a marked decrease in ODC activity, which was paralleled by a decrease in a alpha-DL-difluoromethyl[3,4-3H]ornithine (DFMO)-binding protein of Mr approx. 53,000, which is precipitable with anti-ODC antibody. Calculation of DFMO binding per unit of activity showed no change in the specific activity of the enzyme. We identified [35S]methionine-labelled peptides corresponding to ODC by immunoprecipitation of radiolabeled whole cell proteins. Only one protein was precipitated, of Mr approx. 53 000, which co-migrated with the DFMO-binding protein. Immunoprecipitation of radiolabelled proteins from cells incubated in the presence of exogenous ornithine indicated that the observed activity decrease was not due to an inhibition of ODC protein synthesis. Analysis of immunoprecipitable ODC protein from cells that had been pulse-labelled with [35S]methionine, and then treated for 5 h with 100 microM-ornithine, -putrescine or -spermidine, revealed a distinct disappearance of labelled ODC protein after restoration of intracellular polyamine pools. No detectable turnover of ODC was observed in the absence of exogenous polyamine treatment. These data support the hypothesis that ODC protein, and subsequent activity, is regulated by intracellular polyamine content through mechanisms that influence turnover of the enzyme.

Altered polyamine metabolism in Chinese hamster cells growing in a defined medium.

Chinese hamster cells (line CHO) maintained in McCoy's 5A medium (modified) supplemented with insulin (10 micrograms/ml), transferrin (5 micrograms/ml), and ferrous sulfate (1.1 microgram/ml) proliferate at rates similar to cultures growing in the McCoy's medium supplemented with 10% fetal bovine serum. Colony-forming ability is similar in cultures supplemented with either serum or the combination of growth factors. By 6 hours after replacement of serum with growth factors, ornithine decarboxylase (ODCase) activity increases, reaching a maximum value by 24 hours after serum replacement. This maximum is cell density dependent and can exceed a 30-fold increase over enzyme activity in cultures supplemented with serum. The increased enzyme activity is due to a decrease in the turnover rate of the enzyme, based on protein synthesis inhibition studies, and an accumulation of active enzyme molecules rather than an activation of existing molecules, since the catalytic activity of ODCase, determined using the radiolabeled form of alpha-difluoromethylornithine (an enzyme-activated, irreversible inhibitor of ODCase) in concert with supplements. Intracellular putrescine and spermidine levels are substantially decreased when cultures are maintained in medium supplemented with insulin, transferrin, and ferrous sulfate, rather than serum, which is the sole source of exogenous ornithine. Titration of cultures growing in the defined medium with ornithine leads to a decrease in ODCase activity and an increase in intracellular putrescine and spermidine levels. Putrescine- and spermidine-dependent S-adenosyl-L-methionine decarboxylase activities are similar in cultures maintained in either medium. These data demonstrate that some, but not all, aspects of polyamine biosynthesis are affected by the availability of ornithine, the first substrate in the pathway.

Activation of ornithine decarboxylase in monolayer cells treated with trypsin.

When monolayer Chinese hamster cells are treated with trypsin for short periods of time, ornithine decarboxylase (ODCase) activity increases two- to fourfold. This increase can be blocked by aprotinin, a protease inhibitor, and is not observed when cultures are dislodged from substrate mechanically prior to contact with exogenous trypsin. The trypsin-induced increase in ornithine decarboxylase activity is not due to degradation of enzyme or inhibitor molecules or to new enzyme synthesis. Immunoprecipitable protein, radiolabeled with [3H]alpha-difluoromethylornithine in vitro, is the same molecular weight in cells harvested with or without trypsin. Protein-bound levels of this specific enzyme-activated irreversible inhibitor of ornithine decarboxylase are unchanged by trypsin treatments that increase enzyme activity. Trypsin treatment of rat embryonic fibroblasts, transformed by a temperature-sensitive mutant of Rous sarcoma virus, increases ODCase activity in cells growing at the nonpermissive, but not at the permissive, temperature for the transformed phenotype. These results suggest that ornithine decarboxylase can be activated by exogenous trypsin treatment in a manner that is dependent on cell adhesion properties, which are modified in transformed cells.

Rapid loss of stress fibers in Chinese hamster ovary cells after hyperthermia.

This study was initiated to characterize the effect of hyperthermia (45 degrees) on the distribution of actin stress fibers in Chinese hamster ovary cells using rhodamine-conjugated phalloidin, a probe specific for F-actin. Fluorescent microscopy revealed a rapid loss of stress fibers after immersion in a 45 degrees water bath. After 5-min immersion at 45 degrees, approximately 90% of the cells analyzed did not contain observable stress fibers. Stress fibers were visible after incubation of cells at 37 degrees after heating. The recovery of the appearance of the stress fibers occurred as protein synthesis resumed, and addition of protein synthesis inhibitors following heat treatment blocked the reappearance of these structures. These results support the hypothesis that cytoskeletal components may be a target of hyperthermia, explaining the pleotropic biological effects of heat and, in particular, heat radiosensitization.