Hormone interactions and regulation of PsPK2::GUS compared with DR5::GUS and PID::GUS in Arabidopsis thaliana

The putative pea PINOID homolog, PsPK2, is expressed in all growing plant parts and is positively regulated by auxin, gibberellin, and cytokinin. Here, we studied hormonal regulation of PsPK2::GUS expression compared with DR5::GUS and PID::GUS in Arabidopsis. PsPK2::GUS, DR5::GUS, and PID::GUS expression in Arabidopsis shoots is mainly localized in the stipules, hydathodes, veins, developing leaves, and cotyledons. Unlike DR5::GUS, PsPK2::GUS, and PID::GUS are weakly expressed in root tips. Both DR5::GUS and PsPK2::GUS are induced by different auxins and are more sensitive to methyl indole acetic acid, 4-chloro-indole acetic acid, and α-naphthalene acetic acid than others. GA(3) has no significant effect on GUS activity in DR5::GUS-transformed seedlings compared to the control, but induction by auxin and gibberellin in combination is synergistic. Cytokinin increases auxin transport in Arabidopsis seedlings. Auxin, gibberellin, and cytokinin all increase GUS activity in shoots of PsPK2::GUS transformed plants compared to the control. However, only auxin and gibberellin increase GUS activity in PID::GUS shoots. In conclusion, auxin, gibberellin, and cytokinin positively regulate PsPK2 expression in shoots, but not in roots. Auxin and gibberellin also upregulate AtPIN1 and LEAFY expression, which is similar to PsPIN1 and Uni in pea. With minor exceptions, the orthologous genes from both species are regulated similarly.     

A dimeric RNA quadruplex architecture comprised of two G:G(:A):G:G(:A) hexads,G:G:G:G tetrads and UUUU loops

Using CD and NMR, we determined the structure of an RNA oligomer, r(GGAGGUUUUGGAGG) (R14), comprising two GGAGG segments joined by a UUUU segment. A modified quadruplex structure was observed for r(GGAGGUUUUGGAGG) in solution even in the absence of K(+). An unusually stable dimeric RNA quadruplex architecture formed from two strands of r(GGAGGUUUUGGAGG) at low K(+) concentration is reported here. In each strand of r(GGAGGUUUUGGAGG), two sets of successive turns in the GGAGG segments and turns at both ends of the UUUU loops drive four G-G steps to align in a parallel manner, a core with two stacked G-tetrads being formed. Two adenine bases bind to two edges of one G:G:G:G tetrad through the sheared G:A mismatch augmenting the tetrad into a G:G(:A):G:G(:A) hexad. Thus, one molecule of r(GGAGGUUUUGGAGG) folds into a modified quadruplex comprising a G:G:G:G tetrad, a UUUU double-chain reversal loop and a G:G(:A):G:G(:A) hexad. Two such molecules further associate by stacking through the dimeric hexad-hexad interface with a rotational symmetry. The ribose rings of most nucleotides take S (close to C2'-endo) puckering, which is unusual for an RNA. K(+) can increase the stability of this quadruplex structure; the number of bound K(+) was estimated from the results of the titration experiment. Besides G:G and G:A mismatches, a network of hydrogen bonds including O4'-NH(2) and C-H..O hydrogen bonds, and the extensive base stacking contribute to the high thermodynamic stability of R14. Our results could provide the stereochemical and thermodynamic basis for elucidating the biological role of the GGAGG-containing RNA segments abundantly existing in various RNAs. Relevance to quadruplex-mediated mRNA-FMRP binding and HIV-1 genome RNA dimerization is discussed.     

Experimental demonstration of T:(G:G:G:G):T hexad and T:A:A:T tetrad alignments within a DNA quadruplex stem

A template-based approach was used to design unprecedented architectural motifs into a known DNA framework. The structure formed by the sequence d(GCGGTTGGAT) in 0.1 M Na(+) solution has been determined using molecular dynamics simulations constrained by distance and dihedral restraints derived from NMR experiments. The molecular topology has been previously observed for the sequence d(GCGGTGGAT) (Webba da Silva, M. (2003) Biochemistry 42, 14356-65). Insertion of a single thymine into the double chain reversal formed by the segment GGTGG results in the unprecedented experimental demonstration of a T:(G:G:G:G):T hexad. The bi-stranded hexad results from the pairing alignment of two G(T-G) triads. Each triad results from recognition of the sheared edge of a guanine by the Watson-Crick edge of a thymine of the segment GGTTGG. The alignment is stabilized by base-stacking of the thymine to the sugar pucker of the preceding thymine. The latter is involved in formation of the T:A:A:T tetrad alignment by forming a hydrogen bond with the free amino proton of a Watson-Crick aligned A:A mispair. We have thus established that residues in double chain reversal loops linking juxtaposed tetrads of a quadruplex stem may facilitate formation of yet unknown hydrogen bond alignments. By employing a systematic approach analysis of sequence motifs appearing in double chain reversals, bridging tetrad layers should allow for the prediction of topologies and architectural motifs appearing in biologically relevant genomic regions.     

Leaf lipids of Ribes nigrum: a plant containing 16:3, alpha-18:3, gamma-18:3 and 18:4 fatty acids

The glycerolipid composition of Ribes nigrum (blackcurrant) leaves was determined. The total fatty acid composition was unusual in that alpha-linolenic acid (alpha-18:3) occurred together with cis-7,10,13-hexadecatrienoic acid (16:3) and lower amounts of stearidonic acid (18:4) and gamma-linolenic acid (gamma-18:3). Monogalactosyldiacylglycerol contained the highest proportion of 16:3 with less in digalactosyldiacylglycerol. gamma-18:3 and 18:4 were present in all lipids and 18:4 was always greater than gamma-18:3. The highest percentages of gamma-18:3 and 18:4 were in phosphatidylcholine, but phosphatidylglycerol was particularly low in these acids. In summary, the lipid composition was largely typical of 16:3 plants but there was a minor contribution typical of 18:4 plants. The possibility of three pathways for glycolipid biosynthesis is discussed.     

Quadruplexes of human telomere DNA analogs designed to contain G:A:G:A,G:G:A:A,and A:A:A:A tetrads

Replacement of two to four guanines by adenines in the human telomere DNA repeat dG₃(TTAG₃)₃ did not hinder the formation of quadruplexes if the substitutions took place in the terminal tetrad bridged by the diagonal loop of the intramolecular antiparallel three‐tetrad scaffold, as proved by CD and PAGE in both Na⁺ and K⁺ solutions. Thermodynamic data showed that, in Na⁺ solution, the dG₃(TTAG₃)₃ quadruplex was destabilized, the least by the two G:A:G:A tetrads, the most by the G:G:A:A tetrad in which the adenosines replaced syn‐guanosines. In physiological K⁺ solution, the highest destabilization was caused by the 4A tetrad. In K⁺, only the unmodified dG₃(TTAG₃)₃ quadruplex rearranged into a K⁺‐dependent quadruplex form, none of the multiple adenine‐modified structures did so. This may imply biological consequences for nonrepaired A‐for‐G mutations. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 880–886, 2010.     

Intramolecular higher order packing of parallel quadruplexes comprising a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad of GGA triplet repeat DNA

GGA triplet repeats are widely dispersed throughout eukaryotic genomes and are frequently located within biologically important regions such as gene regulatory regions and recombination hot spot sites. We determined the structure of d(GGA)4 (12-mer) under physiological conditions and founded the formation of an intramolecular parallel quadruplex for the first time. Later, a similar architecture to that of the intramolecular parallel quadruplex was found for a telomere DNA in the crystalline state. Here, we have determined the structure of d(GGA)8 (24-mer) under physiological conditions. Two intramolecular parallel quadruplexes comprising a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad are formed in d(GGA)8. These quadruplexes are packed in a tail-to-tail manner. This is the first demonstration of the intramolecular higher order packing of quadruplexes at atomic resolution. K+ ions, but not Na+ ones, are critically required for the formation of this unique structure. The elucidated structure suggests the mechanisms underlying the biological events related to the GGA triplet repeat. Furthermore, in the light of the structure, the mode of the higher order packing of the telomere DNA is discussed.     

d(CGGTGGT) forms an octameric parallel G-quadruplex via stacking of unusual G(:C):G(:C):G(:C):G(:C) octads

Among non-canonical DNA secondary structures, G-quadruplexes are currently widely studied because of their probable involvement in many pivotal biological roles, and for their potential use in nanotechnology. The overall quadruplex scaffold can exhibit several morphologies through intramolecular or intermolecular organization of G-rich oligodeoxyribonucleic acid strands. In particular, several G-rich strands can form higher order assemblies by multimerization between several G-quadruplex units. Here, we report on the identification of a novel dimerization pathway. Our Nuclear magnetic resonance, circular dichroism, UV, gel electrophoresis and mass spectrometry studies on the DNA sequence dCGGTGGT demonstrate that this sequence forms an octamer when annealed in presence of K(+) or NH(4)(+) ions, through the 5'-5' stacking of two tetramolecular G-quadruplex subunits via unusual G(:C):G(:C):G(:C):G(:C) octads.     

An intramolecular quadruplex of (GGA)(4) triplet repeat DNA with a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad, and its dimeric interaction.

The structure of d(GGAGGAGGAGGA) containing four tandem repeats of a GGA triplet sequence has been determined under physiological K(+) conditions. d(GGAGGAGGAGGA) folds into an intramolecular quadruplex composed of a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad. Four G-G segments of d(GGAGGAGGAGGA) are aligned parallel with each other due to six successive turns of the main chain at each of the GGA and GAGG segments. Two quadruplexes form a dimer stabilized through a stacking interaction between the heptads of the two quadruplexes. Comparison of the structure of d(GGAGGAGGAGGA) with the reported structure of d(GGAGGAN) (N=G or T) containing two tandem repeats of the GGA triplet revealed that although the two structures resemble each other to some extent, the extension of the repeats of the GGA triplet leads to distinct structural differences: intramolecular quadruplex for 12-mer versus intermolecular quadruplex for 7-mer; heptad versus hexad in the quadruplex; and three sheared G:A base-pairs versus two sheared G:A base-pairs plus one A:A base-pair per quadruplex. It was also suggested that d(GGAGGAGGAGGA) forms a similar quadruplex under low salt concentration conditions. This is in contrast to the case of d(GGAGGAN) (N=G or T), which forms a duplex under low salt concentration conditions. On the basis of these results, the structure of naturally occurring GGA triplet repeat DNA is discussed.     

Platelet adhesiveness and aggregation: the collagen:glycosyl, polypeptide:N-acetylgalactosaminyl and glycoprotein:galactosyl transferases of human platelets

Four glycoprotein:glycosyl transferases were identified, purified, and characterized from human platelets. The enzymes present were collagen:glc, collagen:gal, polypeptide:galNAc, and glycoprotein:gal; the fetuin:glcNAc transferase was absent. Each of the 4 transferases was found to be almost exclusively bound to the platelet plasma membrane. Incubation of platelet homogenates without exogenous acceptors yielded no transfer of monosaccharide, indicating the complete lack of endogenous acceptors. These results lead to the interesting speculation that the transferases may not be responsible in the mature platelet for glycoprotein synthesis at all, but rather that they may function for intercellular adhesion and the primary step in hemostasis, the adhesion of collagen to platelets.     

Analysis of Erwinia chrysanthemi EC16 pelE::uidA, pelL::uidA, and hrpN::uidA mutants reveals strain-specific atypical regulation of the Hrp type III secretion system

The plant pathogen Erwinia chrysanthemi produces a variety of factors that have been implicated in its ability to cause soft-rot diseases in various hosts. These include HrpN, a harpin secreted by the Hrp type III secretion system; PelE, one of several major pectate lyase isozymes secreted by the type II system; and PelL, one of several secondary Pels secreted by the type II system. We investigated these factors in E. chrysanthemi EC16 with respect to the effects of medium composition and growth phase on gene expression (as determined with uidA fusions and Northern analyses) and effects on virulence. pelE was induced by polygalacturonic acid, but pelL was not, and hrpN was expressed unexpectedly in nutrient-rich King's medium B and in minimal salts medium at neutral pH. In contrast, the effect of medium composition on hrp expression in E. chrysanthemi CUCPB1237 and 3937 was like that of many other phytopathogenic bacteria in being repressed in complex media and induced in acidic pH minimal medium. Northern blot analysis of hrpN and hrpL expression by the wild-type and hrpL::omegaCmr and hrpS::omegaCmr mutants revealed that hrpN expression was dependent on the HrpL alternative sigma factor, whose expression, in turn, was dependent on the HrpS putative sigma54 enhancer binding protein. The expression of pelE and hrpN increased strongly in late logarithmic growth phase. To test the possible role of quorum sensing in this expression pattern, the expI/expR locus was cloned in Escherichia coli on the basis of its ability to direct production of acyl-homoserine lactone and then used to construct expI mutations in pelE::uidA, pelL::uidA, and hrpN::uidA Erwinia chrysanthemi strains. Mutation of expI had no apparent effect on the growth-phase-dependent expression of hrpN and pelE, or on the virulence of E. chrysanthemi in witloof chicory leaves. Overexpression of hrpN in E. chrysanthemi resulted in approximately 50% reduction of lesion size on chicory leaves without an effect on infection initiation.     

Oka: Behind the Barricades: Kanehsatake: 270 Years of Resistance: Spudwrench: Kahnawake Man: My Name is Kahentiiosta: Acts of Defiance

Oka: Behind the Barricades. 1998. 312 minutes. For more information contact the National Film Board of Canada, 3155 Côte de Lisse Rd., St. Laurent, Quebec H4N 2N4. Series of four videos:
Kanehsatake: 270 Years of Resistance. 1993. 120 minutes. Directed by Alanis Obomsawin .
Spudwrench: Kahnawake Man. 1997. 58 minutes. Directed by Alanis Obomsawin .
My Name is Kahentiiosta. 1995. 30 minutes. Directed by Alanis Obomsawin .
Acts of Defiance. 1992. 104 minutes. Directed by Alec MacLeod .     

Association of DNA quadruplexes through G:C:G:C tetrads. Solution structure of d(GCGGTGGAT)

The structure formed by the DNA sequence d(GCGGTGGAT) in a 100 mM Na(+) solution has been determined using molecular dynamics calculations constrained by distance and dihedral restraints derived from NMR experiments performed at isotopic natural abundance. The sequence folds into a dimer of dimers. Each symmetry-related half contains two parallel stranded G:G:G:G tetrads flanked by an A:A mismatch and by four-stranded G:C:G:C tetrads. Each of the two juxtaposed G:C:G:C tetrads is composed of alternating antiparallel strands from the two halves of the dimer. For each single strand, a thymine intersperses a double chain reversal connecting the juxtaposed G:G:G:G tetrads. This architecture has potential implications in genetic recombination. It suggests a pathway for oligomerization involving association of quadruplex entities through GpC steps.     

Systematics and biogeography of the subantarctic Leptusa (Coleoptera: Staphylinidae: Aleocharinae: Homalotini)

Leptusa atriceps and L. antarctica, flightess representatives of the nearly global genus Leptusa in the remote and widely distributed subantarctic islands, are taxonomically revised. Identity and the widely disjunct distribution of L. atriceps on Falkland, South Georgia, Marion, Crozet and Kerguelen Islands are confirmed. Leptusa antarctica is found to be a complex of five species restricted to the subantarctic islands of New Zealand. Two of these species, L. sparsepunctata and L. nesiotes are here reinstated from synonymy and two others, Leptusa insulae sp. nov. and Leptusa steeli sp. nov., are described as new to science. The monophyly of subantarctic Leptusa species united in the subgenus Halmaeusa was determined by a phylogenetic study of five exemplar homalotine genera and 17 representatives of Leptusa from South America and the Holarctic based on 76 adult morphological characters. Phylogenetic placement of Halmaeusa was not firmly established. Nanoglossa, a temperate South American subgenus of Leptusa was found as a possible sister group to Halmaeusa. The distribution of Halmaeusa species with two to four species that occur sympatrically on Campbell and Auckland Islands, and other species distributed across several remote islands, provides a framework for future biogeographic study of the subantarctic Leptusa.

http://www.zoobank.org/urn:lsid:zoobank.org:act:82509B9D-4BF6-47E1-B6F7-396ED193A0FA; http://www.zoobank.org/urn:lsid:zoobank.org:act:40A3D6FC-27FF-4070-88CA-1B519ABE760A     

Sampling Date,Leaf Age and Root Size: Implications for the Study of Plant C:N:P Stoichiometry

Plant carbon : nitrogen : phosphorus (C:N:P) ratios are powerful indicators of diverse ecological processes. During plant development and growth, plant C:N:P stoichiometry responds to environmental conditions and physiological constraints. However, variations caused by effects of sampling (i.e. sampling date, leaf age and root size) often have been neglected in previous studies. We investigated the relative contributions of sampling date, leaf age, root size and species identity to stoichiometric flexibility in a field mesocosm study and a natural grassland in Inner Mongolia. We found that sampling date, leaf age, root size and species identity all significantly affected C:N:P stoichiometry both in the pot study as well as in the field. Overall, C:N and C:P ratios increased significantly over time and with increasing leaf age and root size, while the dynamics of N:P ratios depended on species identity. Our results suggest that attempts to synthesize C:N:P stoichiometry data across studies that span regional to global scales and include many species need to better account for temporal variation.     

Substrate specificity and sequence preference of G:T mismatch repair: incision at G:T,O6-methylguanine:T,and G:U mispairs in DNA by human cell extracts

Extracts of two human glioma cell lines (lacking O6-methylguanine DNA-methyltransferase) (i.e., A1235 and its alkylation-resistant derivative A1235-MR4) were examined for their ability to execute strand incision at different base mismatches in model (45-bp) DNA. These heteroduplex substrates were of the same sequence except for the presence, at the same site, of one of three mispairs: G:T, O6-methylguanine:T (m6G:T), and G:U. The parental (A1235) extract, when supplemented with ATP and human thymine DNA glycosylase (TDG), acted proficiently on all three substrates, incising immediately 5' to the mismatched thymine or uracil residue. In contrast, the derivative extract, under the same conditions, recognized only the G:U substrate. The activity of the A1235 extract toward the G:T (or m6G:T) substrate was markedly reduced in the absence of ATP, whereas the G:U substrate was incised rapidly by both extracts irrespective of the addition of ATP. These combined data confirm and extend our earlier findings demonstrating that human cells possess two G:T incision activities, one efficient and ATP-dependent and the other inefficient and ATP-independent. The derivative extract lacks the former activity but retains the latter activity. In substrate competition assays, the G:U substrate inhibited the ATP-dependent G:T incision activity to a greater extent than did the G:T substrate itself. Given the well-known substrate preference of TDG for G:U as compared to G:T, this unexpected result implies that TDG may be an integral component of the ATP-dependent G:T incision machinery in human cells. Finally, the base 5' to the mismatched G in the G:T mispair conferred sequence preference on the A1235 extract in the presence of ATP and TDG, with a pyrimidine (especially cytosine) being much favored over a purine. This latter observation suggests that the ATP-dependent G:T incision activity is designed to repair deaminated 5-methycytosine lesions in CpG islands, the methylation of which is linked to control of gene expression.     

W::Neo: a novel dual-selection marker for high efficiency gene targeting in Drosophila

We have recently developed a so-called genomic engineering approach that allows for directed, efficient and versatile modifications of Drosophila genome by combining the homologous recombination (HR)-based gene targeting with site-specific DNA integration. In genomic engineering and several similar approaches, a "founder" knock-out line must be generated first through HR-based gene targeting, which can still be a potentially time and resource intensive process. To significantly improve the efficiency and success rate of HR-based gene targeting in Drosophila, we have generated a new dual-selection marker termed W::Neo, which is a direct fusion between proteins of eye color marker White (W) and neomycin resistance (Neo). In HR-based gene targeting experiments, mutants carrying W::Neo as the selection marker can be enriched as much as fifty times by taking advantage of the antibiotic selection in Drosophila larvae. We have successfully carried out three independent gene targeting experiments using the W::Neo to generate genomic engineering founder knock-out lines in Drosophila.     

Methylglyoxal induces G:C to C:G and G:C to T:A transversions in the supF gene on a shuttle vector plasmid replicated in mammalian cells

We previously reported that the majority of base-pair substitutions induced by an endogenous mutagen, methylglyoxal, were G:C-->T:A transversions and G:C-->A:T transitions in wild-type and nucleotide excision repair (NER)-deficient (uvrA or uvrC) Escherichia coli strains. To investigate the mutation spectrum of methylglyoxal in mammalian cells and to compare the spectrum with those detected in other experimental systems, we analyzed mutations in a bacterial suppressor tRNA (supF) gene in the shuttle vector plasmid pMY189. We treated pMY189 with methylglyoxal and immediately transfected it into simian COS-7 cells. The cytotoxicity and the mutation frequency (MF) increased according to the dose of methylglyoxal. In the mutants induced by methylglyoxal, multi-base deletions were predominant (50%), followed by base-pair substitutions (35%), in which 89% of the substitutions occurred at G:C sites. Among them, G:C-->C:G and G:C-->T:A transversions were predominant. The overall distribution of methylglyoxal-induced mutations detected in the supF gene was different from that for the spontaneous mutations. These results suggest that methylglyoxal may take part in causing G:C-->C:G and G:C-->T:A transversions in vivo.     

Marine nitrogen: Phosphorus stoichiometry and the global N:P cycle

Nitrogen supply is often assumed to limitmarine primary production. A global analysis of totalnitrogen (N) to phosphorus (P) molar ratios shows thattotal N:P is low (<16:1) in some estuarine andcoastal ecosystems, but up to 100:1 in open oceans.This implies that elements other than N may limitmarine production, except in human impacted, estuarineor coastal ecosystems. This pattern may reconcileconflicting enrichment studies, because N additionfrequently increases phytoplankton growth where totalN:P is expected to be low, but P, Fe, or Si augmentphytoplankton growth in waters where total N:P ishigh. Comparison of total N:P stoichiometry betweenmarine and freshwaters yields a model of the form ofthe aquatic N:P cycle.     

C:N:P stoichiometry in soil: is there a “Redfield ratio” for the microbial biomass?

Well-constrained carbon:nitrogen:phosphorus (C:N:P) ratios in planktonic biomass, and their importance in advancing our understanding of biological processes and nutrient cycling in marine ecosystems, has motivated ecologists to search for similar patterns in terrestrial ecosystems. Recent analyses indicate the existence of “Redfield-like” ratios in plants, and such data may provide insight into the nature of nutrient limitation in terrestrial ecosystems. We searched for analogous patterns in the soil and the soil microbial biomass by conducting a review of the literature. Although soil is characterized by high biological diversity, structural complexity and spatial heterogeneity, we found remarkably consistent C:N:P ratios in both total soil pools and the soil microbial biomass. Our analysis indicates that, similar to marine phytoplankton, element concentrations of individual phylogenetic groups within the soil microbial community may vary, but on average, atomic C:N:P ratios in both the soil (186:13:1) and the soil microbial biomass (60:7:1) are well-constrained at the global scale. We did see significant variation in soil and microbial element ratios between vegetation types (i.e., forest versus grassland), but in most cases, the similarities in soil and microbial element ratios among sites and across large scales were more apparent than the differences. Consistent microbial biomass element ratios, combined with data linking specific patterns of microbial element stoichiometry with direct evidence of microbial nutrient limitation, suggest that measuring the proportions of C, N and P in the microbial biomass may represent another useful tool for assessing nutrient limitation of ecosystem processes in terrestrial ecosystems.