Genetic engineering of CHO cells producing human interferon-γ by transfection of sialyltransferases

Natural human interferon- (hIFN-) contains mainly biantennary complex-type sugar chains. We previously remodeled the branch structures of N-glycans on hIFN- in Chinese hamster ovary (CHO) cells by overexpressing UDP-N-acetylglucosamine: 1,6-D-mannoside 1,6-N-acetylglucosaminyltransferase (GnT-V). Normal CHO cells primarily produced hIFN- having biantennary sugar chains, whereas a CHO clone, designated IM4/Vh, transfected with GnT-V, primarily produced hIFN- having GlcNAc1-6 branched triantennary sugar chains when sialylation was incomplete and an increase in poly-N-acetyllactosamine (Gal1-4GlcNAc1-3)n was observed. In the present study, we introduced mouse Gal1-3/4GlcNAc-R 2,3-sialyltransferase (ST3Gal IV) and/or rat Gal1-4GlcNAc-R 2,6-sialyltransferase (ST6Gal I) cDNAs into the IM4/Vh cells to increase the extent of sialylation and to examine the effect of sialyltransferase (ST) type on the linkage of sialic acid. Furthermore, we speculated that sialylation extent might affect the level of poly-N-acetyllactosamine. We isolated four clones expressing different levels of 2,3-ST and/or 2,6-ST. The extent of sialylation of hIFN- from the IM4/Vh clone was 61.2%, which increased to about 80% in every ST transfectant. The increase occurred regardless of the type of overexpressed ST, and the proportion of 2,3- and 2,6-sialic acid corresponded to the activity ratio of 2,3-ST to 2,6-ST. Furthermore, the proportion of N-glycans containing poly-N-acetyllactosamine was significantly reduced (less than 10%) in the ST transfectants compared with the parental IM4/Vh clone (22.9%). These results indicated that genetic engineering of STs is highly effective for regulating the terminal structures of sugar chains on recombinant proteins in CHO cells.     

Cleavage of lambda repressor and synthesis of RecA protein induced by transferred UV-damaged F sex factor

Summary Transfer of a UV-damaged F sex factor to a recipient lysogen induces prophage development. Under these conditions RecA protein synthesis was induced and repressor cleaved, as observed upon direct induction, that is, when the recipient lysogen was directly exposed to UV-light. The efficiency of induction of RecA protein synthesis in recipient bacteria which had received an irradiated F-lac factor was about 80% of that measured upon direct induction. We observed the simultaneous disappearance of repressor and a slight production of cleavage fragments; quantitation by densitometric scanning of the autoradiogram after correction for the efficiency of transfer indicated that 55% of repressor was cleaved. Transfer of UV-damaged Hfr DNA failed to induce RecA protein synthesis. A phage vector carrying oriF, the cloned origin of F plasmid replication, after exposure to UV-light and infection of a recipient lysogen, induced RecA protein synthesis and a moderate but significant cleavage of repressor. Indirect induction by UV-damaged F sex factor or phage oriF resulted in biochemical cellular reactions similar to those observed upon direct induction. LexA repressor that negatively controls RecA protein synthesis appeared more susceptible to cleavage than did repressor.     

Variability in the 11S globulin fraction of seed storage proteins ofHelianthus (Asteraceae)

The seed storage globulins from sixHelianthus and four hybrids were studied using mono and bidimensional gel SDS electrophoresis (+ 2 mercaptoethanol). The polypeptide composition of each subunit was determined. Different pairs are specifically expressed according to the species studied. Three typical patterns were discriminated. All the studied species exhibit five subunits: two of them are expressed in all the species (₁₁ and ₂₂). The subunit corresponding to the ₁₁ pair is present inH. petiolaris and in the three populations ofH. annuus studied. The _2b2 pair is common toH. annuus andH. argophyllus. H. petiolaris presents two specific _2a2 and ₄₄ pairs andH. annuus a specific ₃₃ pair. InH. argophyllus _{11 33} or ₄₄ are never observed but are replaced by ₁₃ and ₃₁ pairs. Some globulins, poorly represented, are of forms but present chains of higher molecular weights (in the range 54–56 kDa). Expressing variations in the banding patterns between these species by the use of a similarity index reveals complete identity between the three populations ofH. annuus. Identity between the twoH. petiolaris studied is also observed.H. annuus andH. argophyllus appear to be closer to each other thanH. petiolaris concerning the seed storage globulins.     

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.     

Polynucleotides and Their Components in the Processes of Aromatic Nucleophilic Substitution: II.1 Nucleophilic Modification of 3′,5′-Bis-O- (α,β,α′,β′-tetrafluoropyrid-γ-yl)thymidine

The interaction of 3,5-bis-O-(,,,-tetrafluoropyrid--yl)thymidine with various nucleophilic reagents was studied to evaluate the possibility of molecular design of new types of nucleic acid analogues using S _NAr reactions. The reactions with morpholine and sodium azide led to the introduction of one and two nucleophilic residues into each of the polyfluorinated pyridine rings. The nucleophilic polycondensation with bifunctional reagents ethylenediamine and hexamethylenediamine depended on the nature of nucleophile and reaction conditions and resulted in the formation of supramolecules containing about five or more than 20 pyrimidine bases.     

Keratella mexicana sp. nov., a new planktonic rotifer from Aguascalientes,Mexico

A new species of Keratella is described from a small reservoir in Aguascalientes, Mexico. The species appears related to K. slacki Brziss, 1963 and K. lenzi Hauer, 1953.     

Genetic analysis of brown planthopper resistance in twenty varieties of rice,Oryza saliva L.

Summary The inheritance of resistance to brown planthopper, Nilaparvata lugens (Stol.), of 20 rice cultivars was studied. Single dominant genes that are allelic to Bph 3 condition the resistance in cultivars Ptb 19, Gangala (Acc. 7733), Gangala (Acc. 15207), Horana Mawee, Kuruhondarwala, Mudu Kiriyal and Muthumanikam. Single recessive genes that are allelic to bph 4 govern the resistance in cultivars Gambada Samba, Heenhoranamawee, Hotel Samba, Kahata Samba, Kalukuruwee, Lekam Samba, Senawee, Sulai, Thirissa and Vellai Illankali. The resistance in Ptb 33, Sudu Hondarwala, and Sinna Sivappu is governed by one dominant and one recessive gene which segregate independently of each other. The dominant resistance genes in these cultivars appear allelic to either Bph 1 or Bph 3. Similarly, the recessive genes in these cultivars seem allelic to either bph 2 or bph 4. Further investigations are needed to conclusively determine the allelic relationships of resistance genes in Ptb 33, Sudu Hondarwala and Sinna Sivappu.     

Triazine dyes as inhibitors and affinity ligands of glycosyltransferases

The triazine dyes: Cibacron Blue 3GA, Reactive Red 120, Reactive Yellow 86, Reactive Green 19, Reactive Blue 4, Reactive Brown 10 inhibited the activity of a purified preparation of 1,6fucosyltransferase (GDP-L-fucose:N-acetyl -glucosaminide 6--L-fucosyltransferase, EC 2.4.1.68) from human blood platelets. Cibacron Blue 3GA and Reactive Red 120 were examined for the nature of the inhibition and both were found to be competitive inhibitors of the enzyme, with K_i=11[emsp4 ]M and 2[emsp4 ]M, respectively. The two dyes inhibited also serum glycosyltransferases: 1,2fucosyltransferase (GDP-L-fucose: -D-galactosyl-R2--L-fucosyltransferase, EC 2.4.1.69), 1,4galactosyltransferase (UDP-galactose: N-acetyl-D-glucosamine 4--D-galactosyltransferase, EC 2.4.1.90) and 1,3N-acetylglucosaminyltransferase (UDP-GlcNAc: 4--D-galactosyl-D-glucose). Cibacron Blue 3GA was a more effective inhibitor of the glycosyltransferases that use UDP-linked sugar donors than Reactive Red 120 while the latter was a stronger inhibitor of the fucosyltransferases that use GDP-linked donor. All four glycosyltransferases could be affinity purified on Cibacron Blue 3GA-Agarose columns. The order of elution of glycosyltransferases from the columns with solutions of 0.25–1.0[emsp4 ]M potassium iodide also depended upon the structure of nucleotide sugar donor, i.e. whether it contained UDP or GDP. Thus, triazine dyes should interact with the sugar donor binding sites of glycosyltransferases. The main advantages of the use of triazine dyes as affinity ligands for isolation of glycosyltransferases are their universal applicability regardless of enzyme specificity, low cost, and insensitivity to high concentration of other proteins present in the solution.     

RAPD analysis of somaclonal variants derived from embryo callus cultures of peach

Peach [Prunus persica (L.) Batsch] regenerants from cv Sunhigh embryo no. 156, regenerants obtained from cv Redhaven embryo no. 30, and two peach cultivars Sunhigh and Redhaven, were screened for polymorphic RAPD (Random Amplified Polymorphic DNA) markers with up to 60 10-mer primers. Although 35 primers produced results with scoreable bands, only 10 of the primers revealed polymorphism for regenerants of embryo no. 156 and cv Sunhigh, and 1 revealed a low level of polymorphism for regenerants of embryo no. 30 and cv Redhaven. This study demonstrates the feasibility of using RAPD markers to identify somaclonal variants of peach and provides evidence for the existence of genetic differences among these variants.Abbreviations PCR Polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP Restriction fragment length polymorphism - CTAB cetyltrimethyl ammonium bromide - PVP polyvinyl pyrolidone - dNTP deoxy-ribonucleotide triphosphate Communicated by R. N. Trigiano     

Plant regeneration and variants from calli derived from immature embryos of diploid barley (Hordeum vulgare L.) and H. vulgare L. x H. bulbosum L. crosses

Summary Plant regeneration from calli was carried out at two locations using several parental genotypes and environments. Selfed immature diploid (VV) barley (Hordeum vulgare) embryos and immature haploid (V) or hybrid (VB) embryos from crosses between H. vulgare and H. bulbosum were used as explants. Golden Promise, Emir and CB 7432 were the best cultivars for plant regeneration, and 15°C tended to be more suitable for plant development than higher temperatures. A total of 844 regenerants were obtained, and over 200 fertile progenies were screened agronomically. Apart from the occurrence of polyploidy and albinism, three variants were identified. One showed maternal inheritance for yellow leaf striping while the other two were controlled by single recessive genes. One of these possessed increased resistance to scald (Rhynchosporium secalis) compared with the donor parent cv Triumph, and one resembled a chlorina mutant.     

Microsatellite markers in the hexaploid Prunus domestica species and parentage lineage of three European plum cultivars using nuclear and chloroplast simple-sequence repeats

In a previous study, cDNA microsatellite markers were described in apricot (Prunus armeniaca L.). Specific PCR primers were designed to amplify the microsatellite-containing regions from genomic DNA in different Prunus species. In the present work, cDNA microsatellite markers were developed in the hexaploid Prunus domestica L. species and polymorphism was ascertained in a segregating plum population. Co-dominant mendelian segregation of alleles was demonstrated and microsatellite polymorphism displayed up to 6 alleles per SSR locus per individual. Parentage lineage of three full-sib European plum cultivars (cv. Cacanska najbolja, Cacanska rana and Cacanska lepotica) was reconstructed by the analysis of the above nuclear SSR markers, completed by four chloroplastic microsatellite loci. The six most informative nuclear loci enabled discrimination between the three Cacak cultivars and unrelated individuals as well as the previously proposed parents, Wangenheim and Pozegaca. Data obtained support previous evidence that these cultivars originated from the Stanley cultivar. However, SSR analysis finally excluded Wangenheim as the other possible parent. Based on the results obtained with nuclear and chloroplast SSR loci, we propose the origin of those three Cacak cultivars in a cross between Stanley as the mother plant and Ruth gerstetter as the pollinator. Furthermore, we demonstrate the utility of these apricot SSR markers for genotype fingerprinting of the hexaploid plum cultivars.     

A single plant technique for field studies of distribution of32P-labelled phosphate between plant and soil pools

Summary ³²P-labelled monocalcium phosphate solution was supplied to the root system of individual wheat plants within a field crop two weeks after emergence. Three levels of carrier P, equivalent to 2.5, 5, and 10 kgP ha^–1 were used. The distribution of³²P between shoots and the soil inorganic and organic P fractions was measured after a further six weeks growth.There was no evidence for the incorporation of³²P-orthophosphate into soil organic P fractions in the wheat rhizosphere in the period between germination and mid-tillering.The suitability of the single plant technique to measure plant available P in field soils was assessed by calculating A values from plants grown in soil witth different fertilizer P histories. There was a significant linear relationship between A values and the amount of soil P extractable with 0.5M NaHCO₃ from the different fertilizer treatments. Although the technique is unlikely to given an absolute value for plant available nutrient, it can provide quantitative data for comparative trials with different forms of fertilizer, methods of fertilizer application or amounts of available soil water. The values obtained should be termed comparative A values or A^c values.     

Glutamine synthetase isoenzymes of Phaseolus vulgaris L.: subunit composition in developing root nodules and plumules

In the legume Phaseolus vulgaris L., glutamine synthetase (GS) (EC.6.3.1.2.) occurs as three cytosolic polypeptides, , and , and a plastidic polypeptide, . This paper describes the subunit composition of active octameric GS isoenzymes from root nodules and plumules using ionexchange high-performance liquid chromatography followed by two-dimensional denaturing gel electrophoresis and Western immunodetection. Root nodules contained four separable GS activities, three of which were composed mainly of cytosolic , / and GS polypeptides, whereas the fourth activity, consisted of plastidic GS polypeptides. The increase in GS activity during nodulation was due largely to the appearance of -containing isoenzymes, and to a lesser extent on the isoenzyme, whereas the -isoenzyme activity remained approximately constant throughout. Plumule GS from imbibed seeds was found to be composed of separate and isoenzymes, but 2 d after germination, plumule GS consisted of a mixture of , / and isoenzymes. The results from both nodules and plumules indicate that different cytosolic GS polypeptides in P. vulgaris are able to assemble into both homo-octameric and heterooctameric isoenzymes. Moreover, the changes in the patterns of isoenzymes observed during nodule development and plumule growth are interpreted to be caused both by temporal changes in the denovo synthesis of the polypeptides and also by their spatial separation in different cell types.Abbreviations 1D, 2D one-, two-dimensional - GS glutamine synthetase - GS_s GS semibiosynthetic activity - GS_t GS transferase activity - IEX-HPLC ion-exchange high-performance liquid chromatography - kDa kilodaltons - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Structural diversity of O-polysaccharides and serological classification of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">garcae</Emphasis> and other strains of genomospecies 4

Novel O-serotypes were revealed among Pseudomonas syringae pv. garcae strains by using a set of mouse monoclonal antibodies specific to the lipopolysaccharide O-polysaccharide. Structural studies showed that the O-polysaccharide of P. syringae pv. garcae NCPPB 2708 is a hitherto unknown linear L-rhamnan lacking strict regularity and having two oligosaccharide repeating units I and II, which differ in the position of substitution in one of the rhamnose residues and have the following structures: I:3)--L Rha (12)-- L Rha (12)--L-Rha-(13)--L Rha (1;II: 2)--L-Rha-(13) -L-Rha-(12)--L-Rha-(13)--L Rha (1.The branched O-polysaccharides of P. syringae pv. garcae ICMP 8047 and NCPPB 588^T have the same L-rhamnan backbone with repeating units I and II and a lateral chain of 14)- or 13)-linked residues of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc). Several monoclonal antibody epitopes associated with the L-rhamnan backbone or the lateral -D-Fuc3NAc residues were characterized.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 777–789.Original Russian Text Copyright © 2004 by Ovod, Zdorovenko, Shashkov, Kocharova, Knirel.     

Role of some epigenetic factors influencing the host suitability of Myzus persicae for the parasitoid Aphidius colemani

When a host is stung by a parasitoid female in the aphid-aphidiid association Myzus persiae Sulzer-Aphidius colemani Viereck, four host responses have been observed: 1) a host develops into a mummy, 2) it dies very soon after it was stung, 3) it develops a cellular immune response, 4) it shows no apparent perturbation subsequent to the sting. The ratios of the responses are clearly determined by the type, the number and the rank of the sting and also by the experience of the female. Some assumptions concerning the cause of the host response are discussed.

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Résumé Le devenir d'un puceron piqué par une femelle parasitoïde dans l'association Myzus persicae Sulzer-Aphidius colemani Viereck est clairement conditionné par différents facteurs associés à la séquence de piqûre. Quatre devenirs ont été régulièrement observés parmi tous les lots d'hôtes piqués; il s'agit d'une mommification classique aboutissant à l'émergence d'un parasitoïde adulte, d'une mort précoce, de l'apparition de profondes modifications anatomiques et physiologiques ou enfin, d'un développement apparemment sain du puceron. Les proportions de ces devenirs varient avec la nature, le nombre, le rang de la piqûre, ainsi qu'avec le passé de la femelle avant l'expérience. Par comparaison avec une piqûre classique, une piqûre peu profonde réduit le nombre de momies formées, mais surtout élimine toute mortalité avancée et favorise le devenir sain. Lorsqu'on augmente de un à trois le nombre de piqûres reçues par l'hôte, la probabilité d'échapper en apparence à un effet quelconque de la piqûre diminue, alors qu'en parallèle, c'est surtout celle de la mommification qui croît. Il existe de plus pour certaines femelles, une relation entre le rang de la piqûre et le devenir de l'hôte. Enfin, si une femelle a dans un premier temps piqué un puceron altéré, près de 90% des pucerons normaux qui sont ensuite piqués évoluent en momies. A partir de ces résultats, certaines hypothèses concernant la cause des différents devenirs sont avancées.

     

Cryptopolyploidy inBunias (Brassicaceae) revisited — A flow-cytometric and densitometric study

The concern of the present analysis is the hypothetical cryptopolyploidy, a concept basically of historical interest only, but discussed again by Battaglia (1996) in a recent treatment of the term and its historical background. Melinossi (1935), while reanalyzing erratic observations on the crucifersBunias erucago andB. orientalis by Jaretzky (1928a), found 2n = 14 in both species but twice the chromosome volume inB. erucago compared withB. orientalis. Melinossi considered cryptopolyploidy inB. erucago, i.e., she discussed pairwise fused chromosomes on a tetraploid basis or endoreduplicated (and thus binemic) chromosomes in this species. Cryptopolyploidy has also been claimed by Pannocchia-Laj (1938) inVinca difformis (Vincaceae). Battaglia (1996) criticized the term cryptopolyploidy because, in his opinion, the genuinely polyploid status of these plants is not hidden (crypto) but phenotypically (from herbarium specimens) recognizable. He coins the term phenopolyploidy, i.e., phenotypic polyploidy disagreeing with the karyotype numerically evalated. We measured genome size ofB. orientalis andB. erucago (both 2n = 14) by Feulgen densitometry and propidium iodide flow cytometry. Surprisingly,B. erucago (the annual species with 2.13 pg, 1 C) turned out to have only 0.81-fold the DNA amount ofB. orientalis (the perennial species with 2.64 pg, 1 C). Therefore, any kind of genetically polyploid status inB. erucago is out of the question. Only speculative significance can be ascribed to the terms cryptopolyploidy and phenopolyploidy.     

sym 18. A novel gene conditioning altered strain specificity in Pisum sativum cv. ‘Sparkle’

An induced mutant of pea Pisum sativum cv. Sparkle forms few nodules with R. leguminosarum bv. viciea from temperate regions, exemplified by strain PRE, but nodulates normally with some rhizobia from Middle East soils, exemplified by strain TOM. The mutant gene is not an allele of sym2, found in the primitive cultivar Afghanistan. Mutant line E54 has a specificity similar to Afghanistan but forms more nodules with temperate strains, especially PF₂ which nodulates Afghanistan only poorly. The new phenotype is conditioned by gene sym18, which can act as recessive or semi-dominant depending on the rhizobial strain. Also sym18 is distinguished from sym 2 by its location on a different linkage group. Sym18 was mapped 9cM from k on linkage group II.     

Correlations between in vivo resistance to Fusarium and in vitro response to fungal elicitors and toxic substances in carnation

Summary With the aim of ascertaining the existance of a correlation between in vivo resistance to Fusarium oxysporum f. sp. dianthi and in vitro response to fungal elicitors and toxic substances, phenylalanine ammonialyase and phytoalexin accumulation, on one hand, and resistance to culture filtrate, on the other, were assayed in in vitro cultures of three susceptible and four resistant Dianthus caryophyllus cultivars. Cultivars showing varying degrees of resistance in vivo either tolerated higher culture filtrate concentrations (Niki) or showed high PAL activity and phytoalexin production when treated with Fusarium elicitor (Duca), or responded positively to both treatments (Mei-Ling, Pulcino). No such responses were shown in tissue cultures of susceptible cultivars. The differential response to the fungal elicitor seemed to be highly specific as genetic differences between cultivars were not observed in tissue cultures treated with other biotic (Phytophthora infestans) and abiotic (HgCl₂) elicitors.Abbreviations FuCWC cell wall components from Fusarium oxysporum f. sp. dianthi race 2 - PhCWC cell wall components from Phytophthora infestans - PAL phenylalanine ammonia-lyase (EC 4.3.1.5)