Genome Analysis of a Natural Hybrid with 2n= 63 Chromosomes in the Genus Elytrigia Desv. (Poaceae) Using the GISH Technique

Abstract: Genomic in situ hybridization (GISH), using genomic DNA probes from Thinopyrum elongatum (E genome, 2 n = 14), Th. bessarabicum (J genome, 2 n = 14), Pseudoroegneria stipifolia (S genome, 2 n = 14), Agropyron cristatum (P genome, 2 n = 28) and Critesion californicum (H genome, 2 n = 14), was used to identify the genome constitution of a natural hybrid population morphologically close to Elytrigia pycnantha and with somatic chromosome number of 2 n = 63. The GISH results indicated the presence of a chromosomal set more or less closely related to the E, P, S and H genomes. In particular, two sets of 14 chromosomes each showed close affinity to the E genome of Th. elongatum and to the P genome of A. cristatum. However, they included 2 and 10 mosaic chromosomes, respectively, with S genome specific sequences at their centromeric regions. Two additional sets (28 chromosomes) appeared to be very closely related to the S genome of Ps. stipifolia. The last genome involved (7 chromosomes) is related to the H genome of C. californicum but includes one chromosome with S genome-specific sequences around the centromere and two other chromosomes with a short interstitial segment also containing S genome related sequences. On a basis of GISH analysis and literature data, it is hypothesized that the natural 9-ploid hybrid belongs to the genus Elytrigia and results from fertilization of an unreduced gamete (n = 42) of E. pycnantha and a reduced gamete (n = 21) of E. repens. The genomic formula SSSSP^SP^SE^SE^SH^S is proposed to describe its particular genomic and chromosomal composition.     

Efficient synthesis of the cyclometalated complex fac-[Rh(ppy)3] (ppy = 2-phenylpyridinato)

A new convenient high-yield synthesis of the tris-cyclometalated complexes fac-[Rh(ppy)₃] (4; ppy = 2-phenylpyridinato) was developed. Complex 4 was prepared in a kind of one-pot synthesis starting from in situ prepared [Rh(acac)(coe)₂] (2) which was heated in refluxing 2-phenylpyridine for a short time. After purification by filtration over alumina, compound 4 was obtained in yields of 65%. Also [Rh(acac)(ppy)₂] (3) was prepared in a similar manner by oxidative addition of Hppy in refluxing toluene in high yields. In contrast to previous findings with the analogous iridium compounds, there was not any hint at the formation of the isomer mer-[Rh(ppy)₃] using similar reaction conditions as applied for iridium. Furthermore the compound [{Rh(μ-Cl)(ppy)₂}₂] (5) was prepared from [{Rh(μ-Cl)(coe)₂}₂] (1) and Hppy in refluxing toluene in nearly quantitative yield.     

RAPD variation in wild populations of four species of the genus Hordeum (Poaceae)

 The genetic variation of 102 natural populations of wild barley growing in Spain was assessed using RAPDs (random amplified polymorphic DNA). The plant material included the annual species H. marinum subsp. marinum (22 populations) and subsp. gussoneanum (14), H. murinum subsp. murinum (7) and subsp. leporinum (35), and the perennial species H. bulbosum (17) and H. secalinum (7). Ten of the tested 64 arbitrary 10-mer primers amplified polymorphic DNA in all taxonomic units. Analyses was performed within and between populations, species and subspecies. The primers gave a total of 250 RAPD products. The level of polymorphism varied between taxonomic units depending on the primers employed and the plant reproductive system. In general, the most variable were the allogamous species H. secalinum and H. bulbosum and the autogamous H. marinum subsp. marinum. Among the amplified bands, 69 (27%) were shared by at least two different taxonomic units. The remaining bands were specific. The results demonstrate differences in the degree of similarity between taxonomic units. Jaccard’s similarity coefficients for interval measure within and between populations were used to produce a cluster diagram using the unweighted pair-group method (UPGMA). The different populations of the species and subspecies of Hordeum fell into three groups. The first group contained the populations belonging to both subspecies of H. marinum, plus those of H. secalinum. The populations of H. marinum subsp. gussoneanum were very closely associated. Those of H. marinum subsp. marinum were grouped in a broad cluster. The second group, occupying the innermost position of the tree, was very closely associated with the populations of both subspecies of H. murinum. The third branch segregated H. bulbosum. A series of RAPD markers were investigated by cleaving the amplified products of the same size with restriction endonucleases that recognize targets of 4- or 6-bp. The production of equivalent fragments following cleavage by the same enzyme would seem to demonstrate their homology in samples from different individuals, populations or taxonomic units. Received: 18 May 1997 / Accepted: 22 August 1997     

Synthesis and solution behavior of the trinuclear palladium(II) unsaturated carboxylate complexes triangle-Pd3[μ-O2CC(R′) = CHMe]6 (R′ = Me, H): X-ray structure of palladium(II) tiglate (R′ = Me)

The first examples of binary palladium(II) derivatives of unsaturated carboxylic acids are reported. It was found that the interaction of Pd₃(μ-OAc)₆ with the ,β-unsaturated 1-methylcrotonic (tiglic) and crotonic acids leads to the corresponding carboxylates of composition Pd₃[μ-O₂CC(R′) = CHMe]₆, where R′ = Me (1) or H (2). The new compounds have been characterized by elemental analysis, solid and solution IR, ¹H and ¹³C NMR, and ESI mass spectrometry. The crystal structure of 1 has been determined. This molecule displays a central Pd₃ cyclic core with Pd–Pd distances of 3.093–3.171 Å. Each Pd–Pd bond is bridged by a pair of carboxylate ligands, one above and the other below the Pd₃ plane, providing a square planar coordination for each Pd atom in an approximate D_3h overall symmetry arrangement. Solution spectroscopic data show that the bridging η¹:η¹:μ₂ interaction of the carboxylates of 1 and 2 is readily displaced, with a change of the ligand to the terminal (η¹) coordination mode.     

Genetic diversity and microgeographic differentiation of Yushan cane (Yushania niitakayamensis; Poaceae) in Taiwan

Yushan cane ( Yushania niitakayamensis ) is distributed in southeast Asia. In Taiwan, the species occurs in mountains 1000–3600 m above sea level. The species appears to spread mainly by rhizomes and flowers only rarely. Nine locations across its distribution range in Taiwan were sampled. Locations at higher altitudes generally consist of grassland and forest undergrowth habitats while those of lower altitudes generally consist of forest undergrowth only. Thus two sampling sites (montane grassland and forest undergrowth) were selected from each location at higher altitudes while only one sampling site was selected from each location at lower altitudes, resulting in a total of 13 sampling sites. Within each sampling site, 20 individual plants were sampled. The results of the cluster analysis and the principal coordinate analysis based on random amplified polymorphic DNA (RAPD) indicated that the populations are generally differentiated according to geographical separation and altitudinal differences that interrupt gene flow. The populations at higher altitudes, where the species is distributed somewhat contiguously, were found to be more similar genetically. Analysis of molecular variance ( AMOVA ) revealed that the among-location, between sampling sites within location, and among individuals within sampling site components accounted for 15.27%, 4.80% and 79.93% of the total variance, respectively. For locations with two sampling sites, two-level AMOVA revealed that the diversities between sampling sites (sun and shade habitats) within locations ranged from 2.91% to 7.99% of the total diversity. Random permutation tests revealed that these diversities were significant, implying that there is microgeographic differentiation due to habitat differences.     

Characterization and function of an E2-17 kDa (UBE2D) in an invertebrate Haliotis diversicolor supertexta

Ubiquitin-conjugating enzymes (UBE2s or E2s) are characterized by the presence of a highly conserved ubiquitin-conjugating (UBC) domain, which predominantly determines the type of ubiquitin chains and directly controls the cellular fate of the substrate. In this study, an E2 homolog was identified and functionally characterized in abalone, which we named ab-UBE2D. The full-length cDNA consists of 1005 bp with an ORF encoding a protein of 147 amino acids. The deduced amino acid sequence shows ab-UBE2D shares conserved UBC domain with other E2 proteins and belongs to class I E2 enzyme family, which are further confirmed by phylogenetic tree analysis. Real-time PCR and western blot analyses showed that ab-UBE2D was ubiquitously expressed in abalone and the expression level of ab-UBE2d was significantly induced by LPS and Poly (I:C). Immunofluorescence microscopy staining demonstrated that native ab-UBE2D was mainly distributed in the cytoplast. Ubiquitination assay showed that ab-UBE2D had ubiquitin conjugating activity to form the enzyme-(Ub)n conjugates. Taken together, these results strongly suggest that ab-UBE2D is an E2 homolog and it may be involved in the immune response of abalone, Haliotis diversicolor supertexta.     

Further evidence supporting an inner sphere mechanism in the NO reduction of the copper(II) complex Cu(dmp)2 (dmp = 2,9-dimethyl-1,10-phenanthroline)

Described are further studies directed towards elucidating the mechanism of the nitric oxide reduction of the copper(II) model system, Cu(dmp)2(2+) (I, dmp=2,9-dimethyl-1,10-phenanthroline). The reaction of I with NO in methanol results in the formation of Cu(dmp)2+ (II) and methyl nitrite (CH3ONO), with a second order rate constant kNO=38.1 M-1 s-1 (298K). The activation parameters for this reaction in buffered aqueous medium were measured to be DeltaH(double dagger)=41.6 kJ/mol and DeltaS(double dagger)=-82.7 kJ/mol deg. The addition of azide ion (N3-) as a competing nucleophile results in a marked acceleration in the rate of the copper(II) reduction. Analysis of the kinetics for the NO reduction of the bulkier Cu(dpp)(2)2+ (IV, dpp=2,9-diphenyl-1,10-phenanthroline) and the stronger oxidant, Cu(NO2-dmp)2(2+) (V, NO2-dmp=5-nitro-2,9-dimethyl-1,10-phenanthroline), gave the second order rate constants kNO=21.2 and 29.3 M-1 s-1, respectively. These results argue against an outer sphere electron transfer pathway and support a mechanism where the first step involves the formation of a copper-nitrosyl (Cu(II)-NO or Cu(I)-NO+) adduct. This would be followed by the nucleophilic attack on the bound NO and the labilization of RONO to form the nitrite products and the cuprous complex.     

Thermodynamic and structural behaviour of equimolar POPC/CnE4 (n = 8, 12, 16) mixtures by sorption gravimetry, 2H NMR spectroscopy and X-ray diffraction

The hydration behaviour of equimolar mixtures of phospholipids and nonionic surfactants with different chain length was investigated by gravimetric sorption, NMR spectroscopy and X-ray diffraction. At the most hydration degrees investigated, the incorporation of nonionic surfactants in a phospholipid bilayer leads to an increase of the hydrophilicity, which can be shown by the presence of excess hydration. The increased hydrophilicity could be explained by the excavation of additional water binding sites due to the “dilution” of the dipole field of the phospholipid bilayer. Another related contribution arises from the increase of the accessible surface area due to the increase of gauche conformers that result from the steric mismatch when surfactants are incorporated into the phospholipid matrix. ²H NMR spectroscopy was used to determine the quadrupolar splitting representing a measure of the order state of water. The swelling behaviour could be assessed by small-angle X-ray diffraction. ³¹P NMR spectroscopy was applied for the assignment of phase structures to the respective hydration range.     

A novel form of intercalation involving four DNA duplexes in an acridine-4-carboxamide complex of d(CGTACG)(2)

The structures of the complexes formed between 9-amino-[N-(2-dimethyl-amino)butyl]acridine-4-carboxamide and d(CG^5BrUACG)₂ and d(CGTACG)₂ have been solved by X-ray crystallography using MAD phasing methodology and refined to a resolution of 1.6 Å. The complexes crystallised in space group C222. An asymmetric unit in the brominated complex comprises two strands of DNA, one disordered drug molecule, two cobalt (II) ions and 19 water molecules (31 in the native complex). Asymmetric units in the native complex also contain a sodium ion. The structures exhibit novel features not previously observed in crystals of DNA/drug complexes. The DNA helices stack in continuous columns with their central 4 bp adopting a B-like motif. However, despite being a palindromic sequence, the terminal GC base pairs engage in quite different interactions. At one end of the duplex there is a CpG dinucleotide overlap modified by ligand intercalation and terminal cytosine exchange between symmetry-related duplexes. A novel intercalation complex is formed involving four DNA duplexes, four ligand molecules and two pairs of base tetrads. The other end of the DNA is frayed with the terminal guanine lying in the minor groove of the next duplex in the column. The structure is stabilised by guanine N7/cobalt (II) coordination. We discuss our findings with respect to the effects of packing forces on DNA crystal structure, and the potential effects of intercalating agents on biochemical processes involving DNA quadruplexes and strand exchanges. NDB accession numbers: DD0032 (brominated) and DD0033 (native).     

Characterization of a novel (±)-abscisic acid metabolite

By application of (±)-abscisic acid and (±)-[1-¹⁴C]-abscisic acid to pea seedlings ( Pisum sativum L. cv. Kleine Rheinländerin), a new abscisic acid metabolite (Pisumic acid, PISA) could be isolated and structurally characterized by combined gas chromatography-mass spectrometry. From data of exact mass determination, it is suggested that the metabolite has the tentative structure of 4'-dihydroabscisic acid with a hydroxylated methyl group at C-6'. That this could be evidence for an alternative pathway of abscisic acid metabolism which was suggested earlier by Walton et al. [Planta 112: 87–90 (1973)] is discussed.     

DNA-binding and molecular mechanics modelling studies of the bulky chiral platinum(II) complex [PtCl2(mepyrr)] (mepyrr = N-methyl-2-aminomethylpyrrolidine)

Detailed studies were carried out on the binding of the enantiomers of [PtCl₂(mepyrr)] (mepyrr = N-methyl-2-aminomethylpyrrolidine) to dG, d(GpG) and a 52-mer oligonucleotide. The pyrrolidine ligand structure was found to be neither sufficiently rigid nor bulky to enforce a single chirality at the exocyclic amine site in this complex, resulting in the presence of diastereomers that complicated the binding studies. Reaction of the (GpG) dinucleotide with R- and S-[PtCl₂(mepyrr)] resulted in formation of four [Pt{d(GpG)}(mepyrr)] isomers for each enantiomer as a consequence of the existence of two orientational isomers and two diastereomers. These isomers formed in different amounts most likely as a consequence of the unequal formation of the diastereomers together with stereoselectivity induced by interactions between the dinucleotide and the mepyrr ligand. The [PtCl₂(mepyrr)] complexes displayed stereoselectivity and enantioselectivity in their reactions with a 52-mer duplex designed to allow formation of only GpG intrastrand adducts. All four bifunctional adducts formed for each enantiomer, providing further evidence of the lack of directing ability of the ligand in formation of the 1,2-intrastrand adduct. Significant amounts of monofunctional species remained in these assays suggesting that the introduction of the methyl substituent to the exocyclic amine inhibited ring-closure to the bifunctional adduct. This was not sufficient to achieve enantiospecificity, but in the case of the R-enantiomer, one of the bifunctional adducts formed in only small amounts.     

Complexity reduction of polymorphic sequences (CRoPS): a novel approach for large-scale polymorphism discovery in complex genomes

Application of single nucleotide polymorphisms (SNPs) is revolutionizing human bio-medical research. However, discovery of polymorphisms in low polymorphic species is still a challenging and costly endeavor, despite widespread availability of Sanger sequencing technology. We present CRoPS as a novel approach for polymorphism discovery by combining the power of reproducible genome complexity reduction of AFLP with Genome Sequencer (GS) 20/GS FLX next-generation sequencing technology. With CRoPS, hundreds-of-thousands of sequence reads derived from complexity-reduced genome sequences of two or more samples are processed and mined for SNPs using a fully-automated bioinformatics pipeline. We show that over 75% of putative maize SNPs discovered using CRoPS are successfully converted to SNPWave assays, confirming them to be true SNPs derived from unique (single-copy) genome sequences. By using CRoPS, polymorphism discovery will become affordable in organisms with high levels of repetitive DNA in the genome and/or low levels of polymorphism in the (breeding) germplasm without the need for prior sequence information.     

Trend in cytotoxic activity of a series of cis-[APtCl2] (A = ethylenediamine methylated at different positions) complexes

The study of a series of cis-[APtCl₂] complexes (A = ethylenediamine, en, methylated at different positions) was carried out to evaluate the effect of different methyl substitutions on the cytotoxic properties of the resulting derivatives. As expected, differentially methylated complexes were found to differ widely in their cytotoxic effects on human cultured ovarian carcinoma cells (A2780). Molecular mechanics (MM) calculations have been performed to assess the relationship between differential diamine methylation and the repulsive energy of the corresponding complexes when interacting with DNA. Compounds that bind DNA at high energetic cost relative to cisplatin, due to the steric hindrance of additional methyl groups, have shown high values for IC₅₀ (concentration inhibiting tumour cell growth by 50%). Semi-quantitative analyses with a DNA electrochemical biosensor confirm that the interaction between cis-[APtCl₂] complexes and ds-DNA deposed onto the electrode is stronger for the non-methylated derivative with respect to the fully methylated congener. In addition, MM calculations were used to investigate the interactions between DNA and cis-[(P-L-A)PtCl₂] complexes [A = en group linked to an antiestrogen-like pharmacophore, P, via a –(CH₂)n– spacer (n = 2, 4, 6, 8 and 10), L].     

Characterization of a complex philadelphia translocation (1p-;9q+;22q-) by gene mapping

Summary Human-Chinese hamster somatic cell hybrids were obtained using circulating leucocytes from a chronic myeloid leukaemia (CML) patient carrying a complex Philadelphia (Ph¹) translocation (1p-; 9q+; 22q-). Hybrid clones which showed segregation of the translocation chromosomes were studied. The chromosome 22 markers ACO2, ARSA, and NAGA segregated with the 1p- derivative; and the chromosome 1 markers UMPK, PGD, and ENO1 segregated with the 9q+ derivative. Hence, molecular evidence has been obtained for the translocation of the distal part of 22q to chromosome 1 and for the translocation of the distal part of 1p to chromosome 9. No conclusions could be drawn either about translocation of chromosome 9 material or about a possible difference in breakpoint in chromosome 22 when compared with six cases of 9;22 translocations similarly studied and previously reported. In addition, a more precise mapping of PGM1 was obtained, the gene being proximal to UMPK and the breakpoint in 1p32.     

Vaccination of sex reversed hybrid tilapia (Oreochromis niloticus × O. aureus) with an inactivated Vibrio vulnificus vaccine

Vibrio vulnificus causes disease in economically important aquaculture raised fish and is an opportunistic human pathogen. This study reports on the isolation of V. vulnificus from diseased hybrid tilapia (Oreochromis niloticus × O. aureus) cultured in a North American water reuse facility. Our objectives were to characterize the isolate using biochemical and molecular methods, develop a disease challenge model, and determine the ability of a formalin inactivated whole-cell vaccine to protect against V. vulnificus. The V. vulnificus isolate recovered was biotype 1, 16S rRNA type B, vcg type C, and vvhA type 2 and caused disease in tilapia held in static salt water (1.5 g/l sea salt). Fish vaccinated with the formalin inactivated whole-cell vaccine responded to vaccination with titers from vaccinated fish ranging from 32 to 64 and titers from non-vaccinated fish ranging from 4 to 8. In two trials, vaccinated tilapia exhibited relative percent survival (RPS) of 73 and 60% following homologous isolate challenge. In two additional trials, vaccinated tilapia exhibited RPS values of up to 88% following challenge with a heterologous isolate; the use of a mineral oil adjuvant enhanced protection. This vaccine may provide an effective means of preventing infections caused by biochemically and genetically diverse V. vulnificus.     

Synthesis,structures and magnetic properties of pentanuclear and trinuclear heterometallic complexes with bended and linear cyano-bridges (tren = tris(2-aminoethyl)amine)

The reaction of [Cu(tren)(OH₂)](ClO₄)₂ with KCN gave a mononuclear complex [Cu(tren)(CN)](ClO₄) (1) (tren = tris(2-aminoethyl)amine). Using 1 as a building block, one pentanuclear compound, [{Cu(tren)(NC)}₄Ni](ClO₄)₆ (2) and two trinuclear complexes, [{Cu(tren)NC}₂Co(tren)](ClO₄)₅ · 2H₂O (3), [{Cu(tren)CN}₂NiL](ClO₄)₄ (4) (L = 3,10-bis(2-hydroxyethyl)-1,3,5,8,10,12-hexaazacyclotetradecane) were prepared and characterized by single crystal X-ray analysis. In 1, Cu(II) atom adopts a distorted trigonal bipyramidal (TBP) geometry. In 2, the Ni(II) atom occupies the center of the pentanuclear compound with a square-planar coordination geometry. In 3, the six-coordinated Co(III) atom presents a distorted octahedral geometry with four nitrogen atoms from tren and two carbon atoms of bridged cyano groups in cis-positions. In 4, the nickel atom is located in an inversion center and coordinated with two [(tren)CuCN]⁺ moieties through cyano-bridging ligands. Magnetic susceptibility measurements of 2–4 show that the magnetic interactions between the heterometallic ions are antiferromagnetical coupling through the cyano bridges with g = 2.25, J = −0.142 cm⁻¹ and J′ = −0.167 cm⁻¹ for 2, g = 2.06, J = −0.094 cm⁻¹ for 3, and g = 2.20, J = −33.133 cm⁻¹ for 4. The correlations between the structures and the J values are discussed.     

Development of novel chloroplast microsatellite markers to identify species in the Agrostis complex (Poaceae) and related genera

We needed a reliable way to identify species and confirm potential interspecific and intergeneric hybrids in a landscape level study of gene flow from transgenic glyphosate-resistant Agrostis stolonifera (Poaceae) to compatible relatives. We developed 12 new polymorphic chloroplast microsatellite markers to aid in identifying species recipient of transgenic pollen both within the Agrostis complex and the related genera Polypogon.     

Phylogenetic analysis of the dimeric alpha-amylase inhibitor sequences from an orthologous region in 21 different genomes of the tribe Triticeae (Poaceae)

Six hundred and thirty gene sequences from 21 different genomes in Triticeae tribe were obtained and subjected to phylogenetic analysis. The sequences showed high homology in both nucleotide sequences and length variation, and had a common conserved cysteine skeleton C–Xn–C–Xn–C–Xn–CC–Xn–C–X–C–Xn–C–Xn–C–Xn–C. The sequences from common wheat formed three clusters; two were close to Aegilops tauschii and Aegilops speltoides sequences, respectively, and the third cluster was complex with sequences from Ae. speltoides, Aegilops searsii, and Aegilops bicornis. Different S genome(s) of Aegilops contributed α-amylase inhibitor loci to polyploid wheat by gene introgression in interspecific hybridizations. No sequence from common wheat was similar to that from einkorn wheat. We conclude that the occurrence of multiple chromosomal translocations or inversions in the different genomes of Triticeae had not dramatically affected the primary structure of dimeric α-amylase inhibitors. The results revealed important information on genome shaping events and processes occurring at the dimeric α-amylase inhibitor genes loci and their bearing on the phylogenetic relationships in the tribe Triticeae (Poaceae).     

Characterization of a novel, ultra-large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86

A novel, ultra-large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86 was purified to homogeneity by ammonium sulfate precipitation and Sephacryl S-300 gel filtration chromatography. The purified xylanosome appeared as a single protein band on the non-denaturing (native) polyacrylamide gel electrophoresis (PAGE) gel with a molecular mass of approximately 1200 kDa. The optimal temperature and pH for xylanase activity was 60 °C and pH 6.0, respectively. The xylanase activity was stable within pH 4.1–10.3. It was stable up to 60 °C at pH 6.0. The xylanosome was highly specific towards oat-spelt xylan, and showed low activity towards corncob powder, but exhibited very low activity towards lichenan, CMC and p-nitrophenyl derivatives. Apparent K_m values of the xylansosome for birchwood, beechwood, soluble oat-spelt and insoluble oat-spelt xylans were 2.5, 3.6, 1.7 and 4.9 mg ml⁻¹, respectively. The main hydrolysis products of birchwood xylan were xylotriose, xylobiose and xylose. Analysis of the products from wheat arabinoxylan degradation by xylanosome confirmed that the enzyme had endoxylanase and debranching activities, with xylotriose, xylobiose, xylose and arabinose as the main degradation products. These unique properties of the purified xylanosome from Streptomyces olivaceoviridis E-86 make this enzymatic complex attractive for biotechnological applications.