Biomedical subjects

R C Gardner

Publications and source records attributed to R C Gardner.

At least 19 recordsLinked to original sources

PubMed2006

A framework linkage map of perennial ryegrass based on SSR markers.

A moderate-density linkage map for Lolium perenne L. has been constructed based on 376 simple sequence repeat (SSR) markers. Approximately one third (124) of the SSR markers were developed from GeneThresher libraries that preferentially select genomic DNA clones from the gene-rich unmethylated portion of the genome. The remaining SSR marker loci were generated from either SSR-enriched genomic libraries (247) or ESTs (5). Forty-five percent of the GeneThresher SSRs were associated with an expressed gene. Unlike EST-derived SSR markers, GeneThresher SSRs were often associated with genes expressed at a low level, such as transcription factors. The map constructed here fulfills 2 definitions of a "framework map". Firstly, it is composed of codominant markers to ensure map transferability either within or among species. Secondly, it was constructed to achieve a level of statistical confidence in the support-for-order of marker loci. The map consists of 81 framework SSR markers spread over 7 linkage groups, the same as the haploid chromosome number. Most of the remaining 295 SSR markers have been placed into their most likely interval on the framework map. Nine RFLP markers and 1 SSR marker from another map constructed using the same pedigree were also incorporated to extend genome coverage at the terminal ends of 5 linkage groups. The final map provides a robust framework with which to conduct investigations into the genetic architecture of trait variation in this commercially important grass species.

Chromosome Mapping↗

PubMed2001

Dark green islands in plant virus infection are the result of posttranscriptional gene silencing.

Dark green islands (DGIs) are a common symptom of plants systemically infected with a mosaic virus. DGIs are clusters of green leaf cells that are free of virus but surrounded by yellow, virus-infected tissue. We report here on two lines of evidence showing that DGIs are caused by posttranscriptional gene silencing (PTGS). First, transcripts of a transgene derived from the coat protein of Tamarillo mosaic potyvirus (TaMV) were reduced in DGIs relative to adjacent yellow tissues when the plants were infected with TaMV. Second, nontransgenic plants coinfected with TaMV and a heterologous virus vector carrying TaMV sequences showed reduced titers of the vector in DGIs compared with surrounding tissues. DGIs also were compared with recovered tissue at the top of transgenic plants because recovery has been shown previously to involve PTGS. Cytological analysis of the cells at the junction between recovered and infected tissue was undertaken. The interface between recovered and infected cells had very similar features to that surrounding DGIs. We conclude that DGIs and recovery are related phenomena, differing in their ability to amplify or transport the silencing signal.

Gene Silencing↗

PubMed2001

A novel family of magnesium transport genes in Arabidopsis.

Magnesium (Mg(2+)) is the most abundant divalent cation in plant cells and plays a critical role in many physiological processes. We describe the identification of a 10-member Arabidopsis gene family (AtMGT) encoding putative Mg(2+) transport proteins. Most members of the AtMGT family are expressed in a range of Arabidopsis tissues. One member of this family, AtMGT1, functionally complemented a bacterial mutant lacking Mg(2+) transport capability. A second member, AtMGT10, complemented a yeast mutant defective in Mg(2+) uptake and increased the cellular Mg(2+) content of starved cells threefold during a 60-min uptake period. (63)Ni tracer studies in bacteria showed that AtMGT1 has highest affinity for Mg(2+) but may also be capable of transporting several other divalent cations, including Ni(2+), Co(2+), Fe(2+), Mn(2+), and Cu(2+). However, the concentrations required for transport of these other cations are beyond normal physiological ranges. Both AtMGT1 and AtMGT10 are highly sensitive to Al(3+) inhibition, providing potential molecular targets for Al(3+) toxicity in plants. Using green fluorescence protein as a reporter, we localized AtMGT1 protein to the plasma membrane in Arabidopsis plants. We suggest that the AtMGT gene family encodes a Mg(2+) transport system in higher plants.

Adenosine Triphosphatases↗

PubMed2001

Evaluation of 1-octen-3-ol, carbon dioxide, and light as attractants for mosquitoes associated with two distinct habitats in North Carolina.

Field studies were conducted in North Carolina to determine the responses of mosquitoes found in salt marsh and inland creek flood plain areas to 1-octen-3-ol (octenol), carbon dioxide (CO2), and light in various combinations with Centers for Disease Control (CDC) light traps. Over 56,000 adult mosquito specimens of 12 species in 4 genera were collected in the salt marsh. They exhibited a general response pattern of octenol + CO2 + light > CO2 + light = octenol + CO2 > octenol + light > octenol alone. Significantly, more Aedes sollicitans, Ae. taeniorhynchus, Anopheles bradleyi, and Culex salinarius were attracted to octenol + CO2 + light than to CO2 + light. Over 19,000 specimens of 24 species in 7 genera were collected in the inland creek flood plain. Although the response patterns to the attractants were similar to those in the salt marsh area, there was no significant difference between octenol + CO2 + light and CO2 + light. Aedes vexans, An. crucians, and An. punctipennis were attracted nearly equally to these two attractant combinations. These studies demonstrate that responses to combinations of these attractants are species specific. However, different combinations of attractants can significantly increase the collection of targeted species important in arbovirus transmission. The use of these combinations would be very beneficial in mosquito-borne virus surveillance studies. The use of octenol by itself or in conjunction with light was found the least useful for collecting mosquitoes in both habitats.

Aedes↗

PubMed2000

Rotavirus VP6 expressed by PVX vectors in Nicotiana benthamiana coats PVX rods and also assembles into viruslike particles.

The rotavirus major inner capsid protein (VP6) has been expressed in Nicotiana benthamiana plants using vectors based on potato virus X (PVX). VP6 was expressed either as a fusion with the PVX coat protein or from an additional subgenomic promoter inserted to enable both VP6 and PVX coat protein to be expressed independently. Both approaches yielded VP6, which retained the ability to form trimers. VP6 expressed from the subgenomic promoter assembled into paracrystalline sheets and tubes. Expression as a fusion protein yielded PVX rods that presented an external "overcoat" of VP6, but unexpectedly, some rotavirus protein also assembled into icosahedral viruslike particles (VLPs). The assembly of viral protein into VLPs suggests that prior display of VP6 on the flexuous PVX rod facilitates the subsequent assembly of VP6 into stable icosahedral particles.

Antigens, Viral↗

PubMed2000

Riding the Ice Age El Nino? Pacific biogeography and evolution of Metrosideros subg. Metrosideros (Myrtaceae) inferred from nuclear ribosomal DNA.

Metrosideros subg. Metrosideros (Myrtaceae) comprises approximately 26 species distributed widely across the Pacific basin. They occur on the ancient Gondwanan landmasses of New Zealand and New Caledonia, as well as on the volcanic islands of the remote Pacific, from Melanesia to tropical Polynesia and the Bonin Island. Phylogenetic analysis based on nuclear ribosomal DNA spacer sequences from all named species showed Metrosideros umbellata of New Zealand as basal in the subgenus, with the remaining species falling into three monophyletic clades. One includes the seven New Caledonian species together with three daughters in western Oceania that probably dispersed during the mid/late Tertiary. A second contains six taxa located in east Melanesia and Samoa that may also have arisen from a mid/late Tertiary dispersal, in this instance from New Zealand. The third includes three New Zealand endemics along with all of the taxa in remote Polynesia and accounts for much of the total range of the subgenus. These dispersed taxa in Polynesia either are identical to the New Zealand species Metrosideros excelsa or differ by a single nucleotide change. We suggest that they are all derived from a Pleistocene dispersal out of New Zealand. A relatively recent dispersal is surprising, given that this wind-dispersed genus has occupied New Zealand for much of the Tertiary and that some of the islands in remote Polynesia date to at least the Miocene. We attribute this dramatic range expansion to climate change-specifically changes in wind flow patterns-in the southern hemisphere during worldwide glaciation.

Base Sequence↗

PubMed2000

Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells.

Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The K(i) values for the best inhibitors were in the range 10-100 nM with suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3xK(i)). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFkappaB activation with IC(50) values comparable to their K(i) values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells.

Animals↗

PubMed2000

Proteasome inhibitors: from in vitro uses to clinical trials.

Proteasomes are multicatalytic proteinase complexes which play a central role in intracellular protein degradation. They catalyse key events in cell cycle regulation and in the activation of the transcription factor NFkappaB. Proteasome inhibitors have been useful for the characterization of proteasome catalytic components and in the elucidation of proteasome functions in animal cells. Potent small peptide inhibitors of proteasomes also represent a novel approach to the treatment of inflammatory diseases (which involve activation of NFkappaB) and cancer. Such compounds have recently been shown to be effective in a variety of animal models, and at least one is currently in use in clinical trials.

Animals↗

PubMed2000

Residues of spinosad in meat, milk, and eggs.

Spinosad is an insect control agent that is derived from a naturally occurring soil bacterium and has a high level of activity against insects that infest a variety of crops. Dairy and poultry feeding studies were conducted to determine the magnitude of spinosad residues in animal products that would result from the consumption of typical feed commodities containing residues of spinosad. Dairy cows were dosed for 28 days with spinosad at rates equivalent to 0, 1, 3, and 10 microg/g in the diet. Chicken hens were dosed for 42 days with spinosad at rates equivalent to 0, 0.1, 0.3, 1, and 5 microg/g in the diet. Milk, eggs, and tissue samples were analyzed by high-performance liquid chromatography and/or immunoassay methods. Spinosad residues occurred in all of the sample types but were lowest in eggs, skim milk, and lean meat and were highest in the fat.

Animals↗

PubMed2000

MDH1: an apple homeobox gene belonging to the BEL1 family.

Differential display was used to isolate genes differentially expressed early in fruit development of apple (Malus domestica Borkh.). This approach resulted in the isolation of MDH1, a homeobox gene with a homeodomain similar to that of BELL1 (BEL1), which is involved in regulation of ovule development in Arabidopsis. However, outside the homeodomain MDH1 is quite different from BEL1. In apple, MDH1 mRNA was predominantly found in flowers, expanding leaves and expanding fruit. In pre-anthesis flowers, in situ hybridization showed that MDH1 mRNA accumulated in ovules. To further investigate the function of this new homeobox gene, MDH1 was transformed into Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. The transgenic Arabidopsis plants showed dwarfing, reduced fertility and changes in carpel and fruit (silique) shape. The size and shape of the cells in the transgenic fruit was irregular. Both the transgenic phenotypes in Arabidopsis and the expression pattern of this gene in apple are consistent with the idea that MDH1 is likely to play an important role in control of plant fertility.

Amino Acid Sequence↗

PubMed2000

Genetic analysis and conservation of 31 surviving individuals of a rare New Zealand tree, Metrosideros bartlettii (Myrtaceae).

Metrosideros bartlettii (Myrtaceae) is a distinctive and extremely rare tree, endemic to New Zealand, first discovered in 1975. Prior to this study, a total of 19 adult individuals of the species had been reported; these are located in three small forest remnants in the far north of the North Island of New Zealand. Here we describe a total of 31 adult M. bartlettii at the three sites, including 12 individuals newly discovered by us. We analyse the genetic diversity of the species, using microsatellites to examine the chloroplast genome and amplified fragment length polymorphisms (AFLPs) to monitor nuclear variation. The results clearly demonstrate that M. bartlettii is a unique species, distinct from its two closest relatives M. robusta and M. excelsa. Analysis of genetic diversity within the 31 remaining individuals of M. bartlettii showed an average heterozygosity (< H >) of 0.18 and a proportion of polymorphic genes (< P >) of 0.44. Population structure, as shown by 286 AFLP loci, varied between the three geographical sites; the site with fewest individuals, containing two trees, showed some separation from the populations at the other two locations. These two latter sites, by contrast, had highly overlapping AFLP population diversity profiles. The implications of these results for conservation of the species are discussed.

Conservation of Natural Resources↗

PubMed2000

Expression of aluminum-induced genes in transgenic arabidopsis plants can ameliorate aluminum stress and/or oxidative stress.

To examine the biological role of Al-stress-induced genes, nine genes derived from Arabidopsis, tobacco (Nicotiana tabacum L.), wheat (Triticum aestivum L.), and yeast (Saccharomyces cerevisiae) were expressed in Arabidopsis ecotype Landsberg. Lines containing eight of these genes were phenotypically normal and were tested in root elongation assays for their sensitivity to Al, Cd, Cu, Na, Zn, and to oxidative stresses. An Arabidopsis blue-copper-binding protein gene (AtBCB), a tobacco glutathione S-transferase gene (parB), a tobacco peroxidase gene (NtPox), and a tobacco GDP-dissociation inhibitor gene (NtGDI1) conferred a degree of resistance to Al. Two of these genes, AtBCB and parB, and a peroxidase gene from Arabidopsis (AtPox) also showed increased resistance to oxidative stress induced by diamide, while parB conferred resistance to Cu and Na. Al content of Al-treated root tips was reduced in the four Al-resistant plant lines compared with wild-type Ler-0, as judged by morin staining. All four Al-resistant lines also showed reduced staining of roots with 2',7'-dichloro fluorescein diacetate (H(2)DCFDA), an indicator of oxidative stress. We conclude that Al-induced genes can serve to protect against Al toxicity, and also provide genetic evidence for a link between Al stress and oxidative stress in plants.

Aluminum↗

PubMed1999

Cloning and expression of a wheat (Triticum aestivum L.) phosphatidylserine synthase cDNA. Overexpression in plants alters the composition of phospholipids.

We describe the cloning of a wheat cDNA (TaPSS1) that encodes a phosphatidylserine synthase (PSS) and provides the first strong evidence for the existence of this enzyme in a higher eukaryotic cell. The cDNA was isolated on its ability to confer increased resistance to aluminum toxicity when expressed in yeast. The sequence of the predicted protein encoded by TaPSS1 shows homology to PSS from both yeast and bacteria but is distinct from the animal PSS enzymes that catalyze base-exchange reactions. In wheat, Southern blot analysis identified the presence of a small family of genes that cross-hybridized to TaPSS1, and Northern blots showed that aluminum induced TaPSS1 expression in root apices. Expression of TaPSS1 complemented the yeast cho1 mutant that lacks PSS activity and altered the phospholipid composition of wild type yeast, with the most marked effect being increased abundance of phosphatidylserine (PS). Arabidopsis thaliana leaves overexpressing TaPSS1 showed a marked enhancement in PSS activity, which was associated with increased biosynthesis of PS at the expense of both phosphatidylinositol and phosphatidylglycerol. Unlike mammalian cells where PS accumulation is tightly regulated even when the capacity for PS biosynthesis is increased, plant cells accumulated large amounts of PS when TaPSS1 was overexpressed. High levels of TaPSS1 expression in Arabidopsis and tobacco (Nicotiana tabacum) led to the appearance of necrotic lesions on leaves, which may have resulted from the excessive accumulation of PS. The cloning of TaPSS1 now provides evidence that the yeast pathway for PS synthesis exists in some plant tissues and provides a tool for understanding the pathways of phospholipid biosynthesis and their regulation in plants.

Amino Acid Sequence↗

PubMed1999

Acquisition of aluminum tolerance in Saccharomyces cerevisiae by expression of the BCB or NtGDI1 gene derived from plants.

Eleven aluminum stress-induced genes derived from plants (wheat, Arabidopsis and tobacco) were introduced into Saccharomyces cerevisiae to test if expression of these genes confers Al tolerance. Al sensitivity tests showed that expression of two genes, either an Arabidopsis gene for blue copper binding protein (BCB), or a tobacco gene for the GDP dissociation inhibitor (NtGDI1), conferred Al tolerance. Determinations of total content and localization of Al ions in these transformants suggested that the BCB gene product functions in restricting Al uptake, while expression of the NtGDI1 gene promotes release of Al ions after uptake.

Aluminum↗

PubMed1999

Genes expressed in Pinus radiata male cones include homologs to anther-specific and pathogenesis response genes.

We describe the isolation and characterization of 13 cDNA clones that are differentially expressed in male cones of Pinus radiata (D. Don). The transcripts of the 13 genes are expressed at different times between meiosis and microspore mitosis, timing that corresponds to a burst in tapetal activity in the developing anthers. In situ hybridization showed that four of the genes are expressed in the tapetum, while a fifth is expressed in tetrads during a brief developmental window. Six of the seven cDNAs identified in database searches have striking similarity to genes expressed in angiosperm anthers. Seven cDNAs are homologs of defense and pathogen response genes. The cDNAs identified are predicted to encode a chalcone-synthase-like protein, a thaumatin-like protein, a serine hydrolase thought to be a putative regulator of programmed cell death, two lipid-transfer proteins, and two homologs of the anther-specific A9 genes from Brassica napus and Arabidopsis. Overall, our results support the hypothesis that many of the reproductive processes in the angiosperms and gymnosperms were inherited from a common ancestor.

Acyltransferases↗

PubMed1999

A set of conserved PCR primers for the analysis of simple sequence repeat polymorphisms in chloroplast genomes of dicotyledonous angiosperms.

Short runs of mononucleotide repeats are present in chloroplast genomes of higher plants. In soybean, rice, and pine, PCR (polymerase chain reaction) with flanking primers has shown that the numbers of A or T residues in such repeats are variable among closely related taxa. Here we describe a set of primers for studying mononucleotide repeat variation in chloroplast DNA of angiosperms where database information is limited. A total of 39 (A)n and (T)n repeats (n > or = 10) were identified in the tobacco chloroplast genome, and DNA sequences encompassing these 39 regions were aligned with orthologous DNA sequences in the databases. Consensus primer pairs were constructed and used to amplify total genomic DNA from a hierarchical set of angiosperms. All 10 primer pairs generated PCR products from members of the Solanaceae, and 8 of the 10 were also functional in most other angiosperm species. Levels of interspecific polymorphism within the genera Nicotiana, Lycopersicon (both Solanaceae), and Actinidia (Actinidiaceae) proved to be high, while intraspecific variation in Nicotiana tabacum, Lycopersicon esculentum, and Actinidia chinensis was limited. Sequence analysis of PCR products from three primer pairs revealed variable numbers of A, G, and T residues in mononucleotide arrays as the major cause of polymorphism in Actinidia. Our results suggest that universal primers targeted to mononucleotide repeats may serve as general tools to study chloroplast variation in angiosperms.

Base Sequence↗

PubMed1998

Protective roles of two aluminum (Al)-induced genes, HSP150 and SED1 of Saccharomyces cerevisiae, in Al and oxidative stresses.

We isolated two yeast cDNA clones whose transcripts are induced by aluminum (Al) metal stress. Partial nucleotide sequencing showed that one is the HSP150 gene encoding a secreted heat shock protein, and the other corresponds to the SED1 gene encoding a putative membrane protein. To clarify the biological functions of these genes, we analyzed the sensitivity of gene-disrupted mutants to Al stress and to oxidative stresses. The Al tests indicated that the HSP150 protein served a basal protective role in Al stress, but SED1 did not; both of the genes had protective roles for oxidative stresses. The results for the HSP150 gene suggest that there is an overlap between Al ion stress, oxidative stress and heat shock stress in yeast.

Aluminum↗

PubMed1998

Overexpression of the Saccharomyces cerevisiae magnesium transport system confers resistance to aluminum ion.

Ionic aluminum (Al3+) is toxic to plants, microbes, fish, and animals, but the mechanism of its toxicity is unknown. We describe the isolation of two yeast genes (ALR1 and ALR2) which confer increased tolerance to Al3+ and Ga3+ ions when overexpressed while increasing strain sensitivity to Zn2+, Mn2+, Ni2+, Cu2+, Ca2+, and La3+ ions. The Alr proteins are homologous to the Salmonella typhimurium CorA protein, a bacterial Mg2+ and Co2+ transport system located in the periplasmic membrane. Yeast strains lacking ALR gene activity required additional Mg2+ for growth, and expression of either ALR1 or ALR2 corrected the Mg(2+)-requiring phenotype. The results suggest that the ALR genes encode the yeast uptake system for Mg2+ and other divalent cations. This hypothesis was supported by evidence that 57Co2+ accumulation was elevated in ALR-overexpressing strains and reduced in strains lacking ALR expression. ALR overexpression also overcame the inhibition of Co2+ uptake by Al3+ ions. The results indicate that aluminum toxicity to yeast occurs as a consequence of reduced Mg2+ influx via the Alr proteins. The molecular identification of the yeast Mg2+ transport system should lead to a better understanding of the regulation of Mg2+ homeostasis in eukaryote cells.

Aluminum↗