Oligomerization of deoxynucleoside-bisphosphate dimers: Template and linkage specificity

Evidence is presented that a poly(U) template selectively favors the oligomerization of the activated, 3–5 pyrophosphate-linked dimer pdAppdAp, in comparison with the 3–3 and 5–5 linked dimers. In the absence of poly(U), the 5–5linked dimer is the most reactive, and chains are formed which are more than 60 monomer units in length.Nucleic Acid-Like Structures V. For the previous paper in this series see Visscher and Schwartz (1988).     

Molecular characterization of β-thalassemia in Hungary

We have identified seven different -thalassemia mutations and one -thalassemia determinant (the Sicilian type) in 32 members of 17 Hungarian families. The most common mutation is the IVS-I-1 (GA) change; its high frequency is comparable to that observed in neighboring Czechoslovakia. Additional mutations are of Mediterranean origin. One rare mutation (initiation codonATGGTG) was identified as an independent mutation because of the absence of known polymorphisms in the -globin gene. One new frameshift at codon 51 (-C) was observed in a single individual; hematological data were as expected for a °-thalassemia heterozygosity.     

Mutant of Escherichia coli with unusual patterns of rpoB,C expression in response to rifampicin and acridine orange

Summary A mutation located near rpoB (89) in E. coli is responsible for unusual patterns of and (but not L7/L12) synthesis in response to the drugs rifampicin and acridine orange.     

Arabidopsis thaliana ‘extra-large GTP-binding protein’ (AtXLG1): a new class of G-protein

Heterotrimeric GTP-binding proteins, composed of , , and subunits, are involved in signal transduction pathways in animal and plant systems. In plants, physiological analyses implicate heterotrimeric G-proteins in ion channel regulation, light signaling, and hormone and pathogen responses. However, only one class of plant G genes has been identified to date. We have cloned a novel gene, Arabidopsis thaliana extra-large GTP-binding protein (AtXLG1). AtXLG1 appears to be a member of a small gene family and is transcribed in all tissues assayed: roots, leaves, stems, flowers, and fruits. The conceptually translated protein from AtXLG1 is 99 kDa, twice as large as typical G proteins. The carboxy-terminal half of the AtXLG1 protein has significant homology to animal and plant G proteins. This region includes a GTP-binding domain, a predicted helical domain, and an aspartate/glutamate-rich loop, which are characteristics of G's. Despite the absence of some of the amino acids implicated in GTP binding and hydrolysis by crystallographic and mutational analyses of mammalian G's, recombinant AtXLGl binds GTP with specificity. The amino-terminal region of AtXLGl contains domains homologous to the bacterial TonB-box, which is involved in energy transduction between the inner and outer bacterial membranes, and to zinc-finger proteins. Given the unique structure of AtXLG1, it will be of interest to uncover its physiological functions.     

Endogenous abscisic acid and 2-trans-abscisic acid in alternate bearing ‘Wilking’ mandarin trees

The relationship of abscisic acid (ABA) and 2-trans-abscisic acid (t-ABA) to alternate bearing has been examined in Wilking mandarin (Citrus reticulata Blanco) trees. Leaves, stems and buds of trees loaded with fruit (on trees) had 4.3, 6.0 and 2.2 fold higher ABA levels than the corresponding organs from off trees. Leaves had higher ABA levels than stems and buds in both on and off trees. t-ABA was non-detectable in Wilking leaf, stem and bud tissue. Amounts of t-ABA not exceeding 40% of the ABA content, were found in Shamouti and Valencia orange buds and in Wilking fruit peel.The elevated levels of ABA in on tree organs may reflect a stress imposed by the fruit overload.     

Genetic analysis of two wheat cultivars, ‘Sonalika’ and ‘WL 711’ for reaction to leaf rust (Puccinia recondita)

Summary Genetic analysis for leaf rust reaction of two widely adapted cultivars, Sonalika and WL 711, has been done using 21 near isogenic Lr lines and rust culture IL004 — avirulent on the two cultivars and all the Lr lines used. The segregation pattern in the F₂ generation indicated the presence of a recessive gene in Sonalika and of a dominant gene in WL 711. These genes in cultivars Sonalika and WL 711 have been identified as Lr 11 and Lr 13, respectively. Gene Lr 13 is no longer effective in WL 711 but it continues to give field resistance in the backgrounds of Chris, Prelude and Thatcher. There has been no significant change in the virulence spectrum of the leaf rust pathogen in India with the release of WL 711. High susceptibility of WL 711 seems to be due to the evolution of more aggressive forms of the pathogen to this cultivar. The gene Lr 11, which behaves as a recessive in Sonalika, was effective against leaf rust when this cultivar was released. The high susceptibility of Sonalika is probably due to an increase in the frequency of race 77 virulent on Lr 11. Lr 11 has shown a dominance reversal in the background of Sonalika. Present results suggest that interaction of resistance genes with the background genotype must be studied for their effective use in breeding programme.     

Characterization of<Emphasis Type="Italic"> Pseudomonas</Emphasis> sp. HK-6 cells responding to explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)

The cellular responses of Pseudomonas sp. HK-6 to explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) have been extensively analyzed in this study. The stress shock proteins, which might contribute to enhancing the cellular resistance to the cytotoxic effect of RDX, were induced at different concentrations of RDX used as a substrate for cell culture of Pseudomonas sp. HK-6. The proteins were identified as 70-kDa DnaK and 60-kDa GroEL by SDS-PAGE and Western blot using the anti-DnaK and anti-GroEL monoclonal antibodies. The stress shock proteins induced by RDX were found to increase in proportion to the RDX concentration used for this work. Analysis of membrane fatty acids of strain HK-6 following exposure to RDX showed that the amounts of dominant lipids 16:1 7c/15:0 iso 2OH, 16:0 and 18:1 7c/9t/12t decreased substantially or were not detected in the cells exposed to RDX, while amounts of lipids 10:0 iso, 14:1 5c/5t and 16:10 methyl increased dramatically. Scanning electron microcopy analyses revealed the presence of perforations and irregular rod shapes with wrinkled surfaces for cells treated with 0.135 mM RDX for 12 h, suggesting that RDX has a substantial cytotoxic impact on cells of strain HK-6.     

Linear differentiation of cereal chromosomes

Summary Using the Giemsa technique of differential staining (the BSG test), we have studies the karyotypes and constructed the idiograms of T. aestivum L. var. Diamant and Chinese Spring, T. Monococcum L. v. hornemannii ssp. roles occidentali georgicum Dek., Aegilops squarrosa L. v. Meeyeri, T. aestivum. The karyotypes of Chinese Spring and Diamant differ drastically both in total structural heterochromatin content and its localization on the nine morphologically homeologous chromosomes. The rest of the twelve pairs of chromosomes showed no morphological similarity. This indicates considerable differences in the phylogeny of the varieties, and also an absence of unique karyotype in T. aestivum.Three chromosomes of Ae. Squarrosa are similar to those of Chinese Spring, yet, on the whole, the chromosomal structural specificity of the diploids studied is so high that we fail to understand the nature of the homology between common wheat and its supposed diploid ancestors.The role of introgression in the evolution of the genomes of Triticum and Aegilops, and also the meaning of the conjugation and BSG tests in resolving the phylogeny, is discussed.     

Agrobacterium-mediated transformation of white clover using direct shoot organogenesis

Summary White clover (Trifolium repens L.) plants from the cultivars Grasslands Huia and Grasslands Tahora have been transformed using Agrobacterium-mediated T-DNA transfer. Transgenic plants regenerated directly from cells of the cotyledonary axil. To transform white clover, shoot tips from 3 day old seedlings were co-cultivated with A. tumefaciens strain LBA4404 carrying the plasmid vector pPE64. This vector contains the neomycin phosphotransferase II gene (nptII) and -glucuronidase reporter gene (gus) both under the control of the CaMV 35S promoter. Kanamycin-resistant plants regenerated within 42 days after transfer onto selective media. Integration of the nptII and gus genes into the white clover genome was confirmed using Southern blotting, and histochemical analysis indicated that the gus gene was expressed in a variety of tissues. In reciprocal crosses between a primary transformant and a non-transformed plant the introduced gus gene segregated as a single dominant Mendelian trait.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - MS Murashige and Skoog - GUS -glucuronidase - X-GLUc 5-bromo-4-chloro-3-indolyl--D-glucuronide - MUG methylumbelliferyl--D-glucuronide - CaMV Cauliflower Mosaic Virus - NPTII neomycin phosphotransferase II - OCS octopine synthase - 4-MU 4-methyl umbelliferone     

Qualitative Analysis of the Carbohydrate Composition of Apolipoprotein H

The specific binding of digoxigenin-labeled lectins to carbohydrate moieties is used to characterize the carbohydrate chains bound to apolipoprotein H. Our results show that apolipoprotein H is rich in sialic acid linked (2–6) to galactose or N-acetylgalactosamine. Sialic acid is not (2–3)-linked to galactose. Galactose is (1–4)-linked to N-acetylglucosamine and (1–3)-linked to N-acetylgalactosamine. High-mannose N-glycan chains are barely detectable. After N-glycosidase F treatment the molecular weight is substantially reduced. The main band is 32,500 daltons. Carbohydrate O-linked chains, which are mainly represented by sialic acid, are (2–6)-linked to galactose or N-acetylgalactosamine. Galactose is also organized in O-linked chains and it is (1–4)-linked to N-acetylglucosamine and (1–3)-linked to acetylgalactosamine. Biochemical analysis of carbohydrate structures reveals that no specific carbohydrate complex is bound to a single isoform.     

The ∈ subunit of the chloroplast ATP synthase of Pisum sativum

In contrast to the well-characterized spinach ( Spinacea oleracea) chloroplast ATP synthase (CF1–CFo), the properties of the chloroplast ATP synthase from pea (Pisum sativum ) have not been as intensively studied. Preliminary data suggested that the regulatory properties of the two enzymes differ. In the absence of activating treatments the ATPase activity of pea thylakoids in the dark was higher than that in spinach thylakoids. When assayed in the presence of sulfite, the MgATPase activity of pea thylakoids was inhibited to a maximum of 67% by tentoxin, indicating that the dark ATPase activity is in part catalyzed by CF1–CFo. The ATPase activity of purified pea CF1 was also higher than that of spinach CF1 in the absence of activating treatments. These differences could result from the different regulatory properties of the pea or subunit or both. The pea subunit was less effective in binding to or inhibiting the ATPase activity of pea o r spinach CF1 deficient in (CF1-). Spinach inhibited the ATPase activity of pea CF1- at lower concentrations than pea . The gene encoding the pea subunit was cloned and over-expressed. Recombinant pea did not restore low proton permeability to spinach thylakoid membranes reconstitituted with spinach CF1-, although pea was effective when tested with pea thylakoids reconstitituted with pea CF1-. These results confirm earlier suggestions that the C-terminal region of is important in -CF1 and -CFo interactions.     

Preliminary crystallographic analysis of the Cks protein p13suc1P90AP92A from Schizosacharromyces pombe

The p13^suc1 is the fission yeast member of the Cks (Cdc28-dependant kinase subunit) family of proteins. The Cks proteins bind to and are required for the function of cyclin-dependant kinase (Cdk) proteins during cell cycle progression in eukaryotic cells. Two conformations of Cks have been detected crystallographically; a compact monomer with the C-terminal fourth -strand inserted into the core of the molecule between strands 2 and 3, and a strand-exchanged dimer where the fourth -strand is inserted into the core of the dimer partner in an equivalent position. There is a highly conserved hinge region consisting of the motif PEP, N-terminal to the fourth -strand. In the monomer this motif constitutes a -turn, while in the dimeric structure it is extended, allowing strand exchange. The mutant protein p13^suc1P90AP92A, in which alanine residues replace both prolines of the turn, provides an opportunity to examine the role of the prolines in this hinge region and how they may allow for the formation of strand-exchanged dimers by Cks proteins. We have expressed and purified this mutant protein. Two millimolar p13^suc1P90AP92A crystallised in 50 mM tris(hydroxymethyl)aminomethane pH 7.5, 30% poly(ethylene glycol) 1500. Diffraction data were collected at room temperature on an MAR345 image plate using Cu K radiation from a Rigaku RU200 rotating-anode generator source to 2.70Å. The crystal has unit cell parameters a=b=75.1 Å, c=34.9 Å, ==90°, =120°. Diffraction data were indexed to the space group P6 and systematic absences 00l indicate a screw axis consistent with P6₃.     

Effect of shape, size, and valency of multivalent mannosides on their binding properties to phytohemagglutinins

Clusters of di-, tri-, and tetra-antennary -D-mannopyranosides were synthesized in good yields based on the coupling of amine-bearing mono- or trisaccharide {Man (16)[Man (13)]Man} haptens to poly-isocyanate or -isothiocyanate tethering cores. The relative binding properties of the resulting multivalent ligands were determined by turbidimetric and solid phase enzyme-linked lectin assays (ELLA) using plant lectins (phytohemagglutinins) Concanavalin A (Con A) and Pisum sativum (pea lectin) having four and two carbohydrate binding sites, respectively. Rapid and efficient cross-linking between tetravalent Con A and mannopyranosylated clusters were measured by a microtiter plate version of turbidimetric analyses. In inhibition of binding of the lectins to yeast mannan, the best tetravalent monosaccharide (30) and trisaccharide (31) inhibitors were shown to be 140 and 1155 times more potent inhibitors than monomeric methyl -D-mannopyranoside against pea lectin and Con A, respectively. Compounds 30 and 31 were thus 35 and 289 fold more potent than the reference monosaccharide based on their hapten contents. As a general observation, the ligands bearing the Man (16)[Man (13)]Man trimannoside structures were found to be more potent inhibitors for Con A than the ligands having single mannoside residues, whereas pea lectin could not discriminate between the two types of ligands.     

Ultrastructural and biochemical study of extracellular matrix vesicles in normal alveolar bone of rats

Summary The immunocharacteristics of androgen target cells in the anterior pituitary of male rats are assessed by a combined thaw-mount autoradiography and immunoperoxidase staining method permitting the simultaneous identification of steroid-hormone target cells and protein or polypeptide hormone-producing cells in the same preparation. After injection of ³H-dihydrotestosterone, nuclear concentration of radioactivity is observed in cells of the anterior lobe, in ectopic cells in the intermediate and posterior lobes as well as in certain pituicytes. The radioactively labeled cells in the anterior lobe are identified immuno-histochemically as gonadotropes and thyrotropes with the use of antiserum to ovine LH or its subunit -LH and bovine -TSH. The results suggest genomic effects of androgen on gonadotropes and thyrotropes as well as pituicytes.Supported by U.S. PHS Grant NS09914     

Molecular characterization of a pea β-1,3-glucanase induced by Fusarium solani and chitosan challenge

-glucanases are prominent proteins in pea endocarp tissue responding to fungal infection. We have cloned and sequenced a partial pea cDNA clone, pPIG312, corresponding to a -1,3-glucanase in pea pods challenged with the incompatible pathogen Fusarium solani f. sp. phaseoli. The insert from the partial pea cDNA was used to probe a genomic library derived from pea leaves of the same cultivar. One of the genomic clones, pPIG4-3, contained the complete coding sequence for a mature -1,3-glucanase protein. The predicted amino acid sequence of the pea -1,3-glucanase has 78% identity to bean -1,3-glucanase, 62% and 60% to two tobacco -1,3-glucanases, 57% to soybean -1,3-glucanase, 51% to barley -1,3-glucanase, and 48% to barley -1,3-1,4-glucanase. Genomic Southern analysis indicates that the pea genome contains only one -1,3-glucanase gene corresponding to the probe used in this study. Accumulation of -1,3-glucanase mRNA homologous with the pPIG312 probe was detected in pea pods within 4 to 8 h after challenge with F. solani f. sp. phaseoli, f. sp. pisi, a compatible strain, or the elicitor, chitosan. In the incompatible reaction, mRNA accumulation remained high for 48h, whereas it rapidly decreased in the compatible reaction. After fungal inoculation of whole pea seedlings, the enhanced mRNA accumulation occurred mainly in the basal region (lower stem and root). This -1,3-glucanase glucanase mRNA was constitutively expressed in the roots of pea seedlings. The sustained levels of -glucanase mRNA expression induced by the incompatible pathogen in the resistance response suggests that the enzyme contributes to the pea plant's general defense.     

Ecological aspects of swidden cultivation among the Andoke and Witoto Indians of the Colombian Amazon

The investigation of crop and soil-crop conditions among Andoke and Witoto cultivators in southeast Colombia is used as a basis for assessing Geertz' (1963) model of swidden cultivation. In this respect, the extent to which maniocdominated swiddens in the study area simulate the structure and composition of the forest climax community is questioned. As Geertz (1963) indicates, an initial nutrient boost for crop cultivation results from the preliminary burning of forest debris, but weed competition, rather than progressive loss of soil fertility, is reported to be the primary cause of abandoning manioc cultivation after 2–3 years. While the Andoke and Witoto crop system remains adaptive at the individual field level, particularly in its constituent species, its fundamental adaptation is considered to be its integration into the broader field and fallow system that juxtaposes crop production with extended periods of forest regeneration.     

Adoptions expérimentales de larves entre des fourmis de genres différents (II):Myrmica laevinodis Nylander etAnergates atratulus Schenck

Résumé Nous avons fait élever des larves d'Anergates atratulus par des ouvrières deMyrmica laevinodis à 22°C. Pour y parvenir, il n'est pas utile de faire hivernerensemble les larves d'Anergates et les ouvrières deMyrmica. La présence de larves autochtones n'empêche pas lesMyrmica d'élever des larves d'Anergates. Dans toutes les expériences lesMyrmica ont été soumises au fridavant de recevoir des larves d'Anergates. Aucune reine deMyrmica n'a été utilisée dans ces expériences.Sur les 64 larves d'Anergates que nous avons utilisées, 38 se sont transformées en imagos. C'est au début de l'adoption et au moment des métamorphoses que périrent la plupart des 26Anergates perdus. Les femelles vécurent en général 2 ou 3 jours et cherchèrent très tôt à quitter le nid natal. Les mâles vécurent 2 à 3 semaines.

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Summary Larvae ofAnergates atratulus were experimentally reared by workers ofMyrmica laevinodis, at 22°C. An overwintering of both larvae ofAnergates and workers ofMyrmica is not necessary for the success of that experiment. The presence of larvae ofMyrmica does not keep theMyrmica from rearing larvae ofAnergates. The workers ofMyrmica have been cooled, in all the experiments, before receiving larvae ofAnergates. No queen ofMyrmica have been used in that experiments.38 of the 64 larvae ofAnergates used became imagos. Most of the 26 lostAnergates died at the beginning of the adoption and during the metamorphosis. The females lived generally 2 or 3 days and tried, very early, to leave their native nest. The males lived 2 or 3 weeks.

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Anergates atratulus Myrmica laevinodis, 22 . bmecme Anergates Myrmica. Myrmica Anergates. Myrmica Anergates. Myrmica . 64 Anergates , 38 . 26 Anergates 2 3 . 2 3 .

     

Development of wheat near-isogenic lines for powdery mildew resistance

Using three Chinese wheat cultivars, Bainong 3217, Beijing 837 and Laizhou 953, as recurrent parents, 33 near-isogenic lines (NILs) carrying 22 powdery mildew resistance genes (Pm1c, Pm2, Pm4b, Pm12, Pm13, Pm16, Pm20, Pm21, Pm23, and 13 undocumented genes) were developed. All NILs had no significant difference to their recurrent parents in the investigated traits of agronomic importance. The results of AFLP analysis indicated Jaccards genetic similarity of the NILs with their recurrent parents varied from 0.96 to 0.98, and confirmed that the NILs had high genetic similarity with their recurrent parents. The resistance to powdery mildew was stably expressed by the relevant NILs. Eleven of the NILs were tested using molecular markers linked to the resistance genes Pm1c, Pm4b, Pm13, Pm21, PmP, PmE, PmPS5A, PmPS5B, PmY39, PmY150, and PmH, and they were all found to carry the targeted genes. The potential application of these NILs in gene discovery is discussed.     

Mutation of a conserved CDK site converts a metazoan Elongation Factor 1Bbeta subunit into a replacement for yeast eEF1Balpha

Elongation factor subunit eEF1B (formerly EF-1 in plants and EF-1 in animals) was identified and cloned in a screen for proteins from pea that interact with a cyclin-dependent kinase (CDK). CDKs are enzymes that regulate progression through meiotic and mitotic cell cycles in eukaryotes. eEF1B and the related protein eEF1B (formerly EF-1' in plants and EF-1 in animals and fungi) can catalyze GTP/GDP exchange on the G-protein eEF1A (formerly EF-1 in plants, animals and fungi) during the elongation phase of protein synthesis in eukaryotes. Recombinant Cdc2 and its native homologues from pea extracts associated both in vitro and in vivo with eEF1B. A Cdc2-cyclin B complex phosphorylated recombinant plant eEF1Bs, but not eEF1B. These interactions between CDK and eEF1B prompted investigations into the in vivo consequences of this relationship. Expression of cDNAs encoding rice or pea eEF1B subunits failed to complement a Saccharomyces cerevisiae mutant deleted for the eEF1B gene, as was previously observed for the human eEF1B. However, replacement of Thr91, the sole consensus CDK phosphorylation site in pea eEF1B, with alanine allowed the pea protein to substitute for eEF1B function in vivo. In addition, this rescued strain was severely cold sensitive, and more sensitive to translational inhibitors than wild-type yeast. Taken together, these results suggest a physiological connection between the cyclin-dependent class of kinases and a translational elongation factor in mitotic cells, and provide the first in vivo evidence that an altered form of eEF1B can serve as the guanine nucleotide exchange factor for eEF1A.Communicated by C. P. Hollenberg