Generation of heteroplastidic Nicotiana cybrids by protoplast fusion: analysis for plastid recombinant types

Summary Protoplasts of a mutant line of Nicotiana tabacum having a maternally-transmitted chlorophyll deficiency were fused with protoplasts of two alloplasmic-male-sterile Nicotiana lines by the donor-recipient technique. In both fusion experiments variegated plantlets were regenerated which were shown to contain cytoplasms of mixed chloroplast nature. This confirms that with the donor-recipient method one can obtain mixed cytoplasms of genetically different chloroplasts. We present a convenient system to assay for genetic recombination between chloroplasts by combining use of several cytoplasmic markers: vis. chlorophyll pigmentation, chloroplast DNA restriction patterns, tentoxin resistance and male sterility. Within the limits of the experiment no recombinant types were recovered.     

Expression and genomic organization of a dinoflagellate gene family

The luciferin-binding protein (LBP) of the dinoflagellate Gonyaulax polyedra is encoded by large gene family with at least two different members. To more fully understand the expression and genomic organization of this gene family, 40 full-length LBP cDNAs were isolated and mapped with the restriction enzymes Xho I, Eco RI, Pvu II and Hind III. All cDNAs isolated could be placed into one of two groups called LBP and LBP. Two LBP group cDNAs were completely sequenced and were found to share 99% identity at both the nucleotide and protein levels. One LBP cDNA was sequenced and was found to share only 86% sequence identity with the LBP group at both the nucleotide and protein levels. Both groups of message appear to be expressed at nearly equal levels since (1) two-dimensional gels of purified LBP show two protein isoforms present in roughly equal amounts and (2) northern blots using group-specific probes suggest that cellular levels of LBP and LBP mRNAs are identical. Genomic Southern blots using group-specific probes suggests that the copy number of both gene groups is very similar and that LBP gene loci are organized as tandem repeats of either LBP or LBP sequences.     

Nuclear Overhauser effect investigation on GM1 ganglioside containingN-glycolyl-neuraminic acid (II3Neu5GcGgOse4Cer)

The conformational properties of the oligosaccharide chain of GM1 ganglioside containingN-glycolyl-neuraminic acid, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-were found to fix the relative orientitation of the three saccharides, while the glycosidic linkage of the terminal -Gal-was found to be quite mobile as the -Gal-(1-3)--GalNAc-disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containingN-acetyl-neuraminic acid and neuraminic acid [1].Abbreviations Ganglioside nomenclature is in accordance with Svennerholm [23] and the IUPAC-IUB Recommendations [24] GM3(Neu5Ac) II³Neu5AcLacCer, -Neu5Ac-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM3(Neu5Gc) II³Neu5GcLacCer, -Neu5Gc-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Ac) II³Neu5AcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Gc) II³Neu5GcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu) II³NeuGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu-(2-3)]--Glc-(1-1)-Cer - GD1a IV³Neu5AcII³Neu5AcGgOse₄Cer, -Neu5Ac-(2-3)--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GalNAc-GD1a IV⁴GalNAcIV³Neu5AcII³Neu5AcGgOse₄Cer, -GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - Neu neuraminic acid - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolyl-neuraminic acid - Cer ceramide     

Presence of carotenoids in Gordius aquaticus (Nematomorpha,Nemathelminthes) specimens from deep wells

By means of columnar and thin-layer chromatography, the presence of carotenoids in Gordius aquaticus L. (Nematomorpha, Nemathelminthes) from deep wells was studied.The investigations revealed the presence of the following carotenoids: -carotene, mutatochrome, -cryptoxanthin, ,-carotene epoxide,lutein, zeaxanthin and astaxanthin ester.     

Nuclear DNA changes,genome differentiation and evolution inNicotiana (Solanaceae)

A survey of 51 species fromNicotiana subgg.Tabacum, Rustica andPetunioides has shown that evolution was accompanied by a five-fold variation in nuclear DNA amounts. This variation, however, was not directly correlated with the changes in chromosome number. Drastic rearrangement of karyotypes is characteristic for the evolution ofNicotiana spp. Significant gain or loss in nuclear DNA has often accompanied such changes, but DNA variation has also occurred without significant changes in karyotype arrangements.—The distribution of nuclear DNA is discontinuous inNicotiana, species cluster into DNA groups with consistently regular increments in the mean DNA amounts. The discontinuities are viewed as steady states in terms of genomic balance and biological fitness.—Changes in the amount of nuclear DNA and in the heterochromatin are compared with the morphological, chromosomal and adaptive changes which accompanied speciation in 14 subgeneric sections. The evolutionary significance of DNA variation is discussed.     

Infloreszenz- und Blütenentwicklung bei der KugeldistelEchinops exaltatus (Asteraceae)

InEchinops the flowers are surrounded by several scales and initiated in an acropetal and spiral succession on a cone-like inflorescence axis (Figs. 1–6). The floral organs originate in the following sequence: petals—stamens—carpels—pappus. The petals arise from a meristematic rim and therefore are already interconnected when they arise as primordia. This sympetalous zone remains rather inconspicuous for a long period, but eventually, the elongated corolla tube is formed through intercalary growth in a ring zone. Thereby, the stamens are moved upwards and form ledges on the corolla tube (Fig. 34). In the inferior ovary the usual zones of the typical angiospermous gynoecium can be distinguished, namely a synascidiate, symplicate and hemisymplicate zone. The ovule is borne on carpellary tissue.

     

Further cytogenetic analyses of the Croatian triploid shallot “Ljutika” (Allium cepa var.viviparum, Alliaceae) and its comparison with the Indian triploid “Pran”

Feulgen and silver-stained karyotypes and meiosis of two triploid viviparous onion forms (Allium cepa var.viviparum), the Croatian Ljutika and the Indian Pran, were comparatively analyzed. The results of chromosome measurements show that Ljutika and Pran are karyologically not identical, although significant similarities were found in the morphology of their chromosomes. Five geographically distant clones of Ljutika showed good agreement in the number and gross morphology of the chromosomes and in the number and position of NORs and interphase nucleoli. Heterotrivalents were predominant in meiosis of Ljutika but a relatively high frequency of higher multivalents together with univalents and bivalents were also observed. The relationship between Ljutika and Pran and their possible origin are discussed.     

Physicochemical and immunological characterization of the type E botulinum neurotoxin binding protein purified fromClostridium botulinum

Type E botulinum neurotoxin is produced byClostridium botulinum along with a neurotoxin binding protein which helps protect the neurotoxin from adversepH, temperature, and proteolytic conditions. The neurotoxin binding protein has been purified as a 118-kDa protein. Secondary structure content of the neurotoxin binding protein as revealed by far-UV circular dichroism spectroscopy was 19% -helix, 50%-sheets, 28% random coils, and 3%-turns. This compared to 22% -helix, 44%-sheets, 34% random coils, and no-turns of the type E botulinum neurotoxin. The complex of the two proteins revealed 25%-helix, 45%-sheets, 27% random coils, and 3%-turns, suggesting a significant alteration at least in the-helical folding of the two proteins upon their interaction. Tyrosine topography is altered considerably (28%) when the neurotoxin and its binding protein are separated, indicating strong interaction between the two proteins. Gel filtration results suggested that type E neurotoxin binding protein clearly complexes with type E neurotoxin. The interaction is favored at lowpH as indicated by an initial binding rate of 8.4 min^–1 atpH 5.7 compared to 4.0 min^–1 atpH 7.5 as determined using a fiber optic-based biosensor. The neurotoxin and its binding protein apparently are of equivalent antigenicity, as both reacted equally on enzyme-linked immunosorbent assay to polyclonal antibodies raised against the toxoid of their complex.     

Control of glycoprotein synthesis: substrate specificity of rat liver UDP-GlcNAc:Manα3R β2-N-acetylglucosaminyl-transferase I using synthetic substrate analogues

UDP-GlcNAc: Man3R 2-N-acetylglucosaminyltransferase I (GlcNAc-T I; EC 2.4.1.101) is the key enzyme in the synthesis of complex and hybrid N-glycans. Rat liver GlcNAc-T I has been purified more than 25,000-fold (M _r 42,000). TheV _max for the pure enzyme with [Man6(Man3)Man6](Man3)Man4GlcNAc4GlcNAc-Asn as substrate was 4.6 µmol min^–1 mg^–1. Structural analysis of the enzyme product by proton nuclear magnetic resonance spectroscopy proved that the enzyme adds anN-acetylglucosamine (GlcNAc) residue in 1–2 linkage to the Man3Man-terminus of the substrate. Several derivatives of Man6(Man3)Man-R, a substrate for the enzyme, were synthesized and tested as substrates and inhibitors. An unsubstituted equatorial 4-hydroxyl and an axial 2-hydroxyl on the -linked mannose of Man6(Man3)Man-R are essential for GlcNAc-T I activity. Elimination of the 4-hydroxyl of the 3-linked mannose (Man) of the substrate increases theK _M 20-fold. Modifications on the 6-linked mannose or on the core structure affect mainly theK _M and to a lesser degree theV _max, e.g., substitutions of the Man6 residue at the 2-position by GlcNAc or at the 3- and 6-positions by mannose lower theK _M, whereas various other substitutions at the 3-position increase theK _M slightly. Man6(Man3)4-O-methyl-Man4GlcNAc was found to be a weak inhibitor of GlcNAc-T I.Abbreviations BSA Bovine serum albumin - Bn benzyl - Fuc, F l-fucose - Gal, G d-galactose - GalNAc, GA N-acetyl-d-galactosamine - Glc d-glucose - GlcNAc, Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man, M d-mannose - mco 8-methoxycarbonyl-octyl, (CH₂)₈ COOOCH₃ - Me methyl - MES 2-(N-morpholino)ethanesulfonate - NMR nuclear magnetic resonance - PMSF phenylmethylsulfonylfluoride - pnp p-nitrophenyl - SDS sodium dodecyl sulfate - T transferase - Tal d-talose - Xyl d-xylose; - {0, 2 + F} Man6 (GlcNAc2Man3) Man4GlcNAc4 (Fuc6) GlcNAc - {2, 2} GlcNAc2Man6 (GlcNAc2Man3) Man4GlcNAc4GlcNAc; M₅-glycopeptide, Man6 (Man3) Man6 (Man3) Man4 GlcNAc4GlcNAc-Asn Enzymes: GlcNAc-transferase I, EC 2.4.1.101; GlcNAc-transferase II, EC 2.4.1.143; GlcNAc-transferase III, EC 2.4.1.144; GlcNAc-transferase IV, EC 2.4.1.145; GlcNAc-transferase V, UDP-GlcNAc: GlcNAc2 Man6-R (GlcNAc to Man) 6-GlcNAc-transferase; GlcNAc-transferase VI, UDP-GlcNAc: GlcNAc6(GlcNAc2) Man6-R (GlcNAc to Man) 4-GlcNAc-transferase; Core 1 3-Gal-transferase, EC 2.4.1.122; 4-Gal-transferase, EC 2.4.1.38; 3-Gal-transferase, UDP-Gal: GlcNAc-R 3-Gal-transferase; blood group i 3-GlcNAc-transferase, EC 2.4.1.149; blood group I 6-GlcNAc-transferase, UDP-GlcNAc: GlcNAc3Gal-R (GlcNAc to Gal) 6-GlcNAc-transferase.     

Isolation of molecular markers for tomato (L. esculentum) using random amplified polymorphic DNA (RAPD)

Summary A new DNA polymorphism assay was developed in 1990 that is based on the amplification by the polymerase chain reaction (PCR) of random DNA segments, using single primers of arbitrary nucleotide sequence. The amplified DNA fragments, referred to as RAPD markers, were shown to be highly useful in the construction of genetic maps (RAPD mapping). We have now adapted the RAPD assay to tomato. Using a set of 11 oligonucleotide decamer primers, each primer directed the amplification of a genome-specific fingerprint of DNA fragments. The potential of the original RAPD assay to generate polymorphic DNA markers with a given set of primers was further increased by combining two primers in a single PCR. By comparing fingerprints of L. esculentum, L. pennellii, and the L. esculentum chromosome 6 substitution line LA1641, which carries chromosome 6 from L. pennellii, three chromosome 6-specific RAPD markers could be directly identified among the set of amplified DNA fragments. Their chromosomal position on the classical genetic map of tomato was subsequently established by restriction fragment length polymorphism (RFLP) linkage analysis. One of the RAPD markers was found to be tightly linked to the nematode resistance gene Mi.     

Nicotiana chloroplast genome

Summary A physical map containing six restriction sites of the Nicotiana tabacum chloroplast genome, together with the BamHI maps of N. tabacum, N. otophora and N. knightiana, and the SmaI maps of N. acuminata, N. plumbaginifolia, N. langsdorffii, N. otophora, N. tabacum, N. tomentosiformis and N. knightiana was constructed. In Nicotiana chloroplast genomes, the most frequently observed variations are point mutations. Deletions are also detected. Most of the observed changes are confined to one area of the large single copy region, which is designated as the hot spot. Based on the evidence obtained from Nicotiana chloroplast genomes, an origin of the inverted repeats in this genus is proposed. We suggest that the inverted repeats represent a vestige of what were once two identical, complete chloroplast genomes joined together in a head-to-head and tail-to-tail fashion, and that deletions generated the current chloroplast genome organization.     

Calling song recognition in the cricket Teleogryllus oceanicus: comparison of the effects of stimulus intensity and sound spectrum on selectivity for temporal pattern

The turning response to surface waves of clawed toads (Xenopus laevis) with an inactivated lateral line was reinvestigated to examine whether sensory systems other than the lateral line (second systems) are involved. Two methods were used to block the lateral line input: selective and reversible inactivation of the lateral line periphery using CoCl₂ or chronic destruction with thermocautery. The time-course of the response recovery (response frequency, turning accuracy and reaction time) was recorded. Following CoCl₂ inactivation 10 out of 13 animals did not respond to surface waves for at least 2 days. The remaining 3 animals gave sporadic turning responses. It is assumed that in these individuals a second system is permanently involved in the detection of surface waves parallel to the lateral line. Five days after the chronic destruction of the lateral line all animals again turned to the centre of surface waves. It is suggested that by this time the second system had become capable of substituting for the missing lateral line input. The response frequency and the accuracy of the turning response of lesioned animals varied considerably among individuals but was always lower than in untreated animals (tested up to 120 days).Abbreviation LED light emitting diode     

Genetic variation detected by use of the M13 “DNA fingerprint” probe in Malus,Prunus, and Rubus (Rosaceae)

Summary Recently, DNA fingerprints have been reported in a wide array of organisms. We used the M13 repeat probe on several genera and species in the angiosperm family Rosaceae. Four apple cultivars could be differentiated when any one of five restriction enzymes was used to analyze minisatellite DNA. Similarly, four individual trees of Prunus serotina (black cherry) exhibited different fingerprints with each of four enyzmes. A total of 14 Rubus (blackberries and raspberries) plants representing four species were investigated with two enzymes. Extensive inter-and intraspecific variation was found. However, some closely growing plants had identical fingerprints, probably due to their being derived through vegetative propagation.     

A study of the HLA-D region in patients with classic Kaposi's sarcoma

The HLA-D region in nine Sardinian patients with classic Kaposi's sarcoma was studied with two restriction enzymes, Eco RI and Eco RV, and two cDNA probes, DR and DQ. A total of 41 polymorphic restriction fragments were identified. One, an 11.5 kb Eco RV DQ fragment, was present in three of the patients but in none of the controls; a second, an 8.0 kb Eco RV DR fragment, was present in six patients and all the controls. No single fragment was identified which was significantly over or under-represented in either group.     

Die Richtungen des Nahrungsflusses im Darmtrakt der Kleinzikade Euscelidius variegatus KBM. (Jassidae)

Zusammenfassung Der Verlauf des Nahrungsflusses im Darmtrakt der Kleinzikade Euscelidius variegatus wird nach Verfütterung von farbstoffhaltiger Nährlösung ermittelt. Es wird der Beweis erbracht, daß die aufgenommene Nahrungsmenge in der Filterkammer geteilt wird und die beiden Anteile den Darmtrakt auf zwei verschiedenen Wegen in Richtung Rektalblase passieren. Ein Anteil der aufgenommenen Nährlösung wird über einen Kurzschlußweg in der Filterkammer sowohl über den Filterkammerdarm als auch über die Kryptonephridien direkt in den Enddarm gepumpt, während die in der Magentasche der Filterkammer verbleibenden Nahrungsanteile über einen langen Verdauungsweg zum After gelangen. Hierbei wird der Magentascheninhalt in den Magen gedrückt. Von dort aus passiert er den Mitteldarm und erreicht über den Enddarm den After. Der Kurzschlußweg und der Verdauungsweg können gleichzeitig benutzt werden. Der Kurzschlußweg wird von der Nahrung jedoch in viel kürzerer Zeit durchströmt als der längere Verdauungsweg.

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The directions of the flow of food in the alimentary trad of the leafhopper Euscelidius variegatus KBM. (Jassidae)

Summary The leafhopper Euscelidius variegatus is fed with synthetic food, coloured with 1% Azorubin-S. Its flow in the alimentary tract has been studied. It has been found that the sucked-in food is divided into two parts in the filter chamber, each taking different way in the alimentary tract for its flow. One part of the food is pumped into the hindgut via the short circuit way going through the filter chamber once over the Filterkammerdarm and also over the kryptonephries. That part of the food, which remains in the pocket of the filter chamber takes the long digestion way to the anus over stomach, midgut and hindgut. Both the ways could be used at the same time. But the food takes much shorter time for its passage through the short circuit way as compared to the time needed for the long digestion way.

     

Utilization of wild relatives in the genetic improvement of Arachis hypogaea L.

Summary Synthetic amphidiploids were established in 32 combinations involving 8 diploid wild species representing both A and B genomes of section Arachis. Bivalent and multivalent associations in the amphidiploids of 7 A genome species confirm that these species have identical genomes. Contrastingly, high bivalent frequencies in amphidiploids involving the A and B genome species suggest that A. batizocoi has a distinct B genome that is partially homologous to the other genome A represented in the rest of the species. Crossability, chromosome pairing and pollen and pod fertility in hybrids between A. hypogaea and amphidiploids have revealed that these amphidiploids can be used as a genetic bridge for the transfer of genes from the wild species into the cultivated groundnut.Submitted as Journal Article No. 530 by International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)     

Oviposition choices by a pre-dispersal seed predator (Hylemya sp.)

Summary Although the importance of pollinators has most often been examined in the evolution of floral characters, seed predators may also play a role in shaping floral evolution. In this study, I examined the role of interplant distance, plant size, and flower morphology on Ipomopsis aggregatás (Polemoniaceae) attractiveness to a pre-dispersal seed predator, Hylemya sp. (Anthomyiidae) and to hummingbird pollinators. The attractiveness of I. aggregata individuals to Hylemya was nonlinearly related to interplant distance in experimental arrays. Clumped and highly dispersed plants were preyed upon more frequently than those at intermediate distances. I found no relationship between interplant distance and visitation rates by hummingbird pollinators in these experimental arrays. However, in natural populations studied, clumped plants were more frequently approached by hummingbirds than those growing more widely dispersed. Display size was unrelated to visitation by Hylemya on inflorescences I clipped and maintained as large, small and control. Display size was also unrelated to the total number of visits by hummingbird pollinators to each of these experimental plants, however large display plants were more likely to be visited first in any given visitation sequence. Of various morphological measurements, corolla length showed the strongest positive correlation with Hylemya egg presence. To the extent that plant spacing and morphology is correlated with pollinator visits and ultimate seed set, Hylemya could be choosing flowers optimally, and playing a role in the evolution of floral traits.     

α-and β-Glycosidases in maize roots

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for -glucosidase, 4.4 for -galactosidase, 6.4 for -glucosidase and 6.0 for -galactosidase. The -glucosidase showed 4-fold higher activity than the -galactosidase. The distribution of the -glucosidase activity was signifcantly different from that of the -galactosidase, -glucosidase and -galactosidase.Abbreviations -Glu -glucosidase - -Gal -galactosidase - -Glu -glucosidase - -Gal -galactosidase     

Cell junctions with funnels in the olfactory mucosa of frogs (Rana temporaria L.)

Summary A characteristic structure in the apical junctional belt of the olfactory epithelium in Rana temporaria is visible in freeze-fracture preparations. This structure is described as a funnel with channel across the junctional belt. It is supposed to represent a possible way for discarding used molecules after stimulation, and to allow the stimulation of free nerve endings in the depth of olfactory epithelia.I wish to thank Prof. C.F. Bardele and Mr. H. Schoppmann for their kind support and technical help