Phylogeny of Cucumis based on isozyme variability and its comparison with plastome phylogeny

Summary An electrophoretic comparison of 29 nuclear-coded enzymes was carried out for 21 Cucumis species, and a phylogeny based on pairwise measurements of the respective genetic distances was computed. This phylogeny was compared to the one based on chlDNA cariation (Perl-Treves and Galun 1985). The two phylogenies were found to share the main dendrogram features; they also agree well with most taxonomic data available on Cucumis. Accordingly, most of the African Cucumis species form a close group (Anguria group — Group A), which is distant from the melon (C. melo), and from a few other distinct species, all of which are far apart from each other. The cucumber (C. sativus) is the most distant species within the genus. Some specific taxonomic implications as well as some general evolutionary problems related to such a parallel investigation of the nuclear genome and the plastome are evaluated.This publication is dedicated to Dr. T. W. Whitaker, with appreciation to his many contributions to genetics and taxonomy of Cucurbitaceae     

Meiotic studies ofElymus nutans andE. jacquemontii (Poaceae,Triticeae) and their hybrids withPseudoroegneria spicata and seventeenElymus species

Hybridizations ofElymus nutans andE. jacquemontii were carried out with one species ofPseudoroegneria (S genome), and 20Elymus species, each containing either of the SH, SY, SYH, or SYW genomes. Chromosome configurations were analysed at metaphase I of the two target taxa and their interspecific hybrids. It is concluded that (i)E. nutans is an allohexaploid containing the SYH genomes, andE. jacquemontii is an allotetraploid having the SY genomes; (ii) the genomic affinity is associated with the geographic distance between the species studied; (iii) minor genomic structural rearrangements have occurred within the hexaploid taxon ofE. nutans.     

Phylogenetic relationships among Cucumis species based on the ribosomal internal transcribed spacer sequence and microsatellite markers

The phylogenetic relationships within the genus Cucumis (a total of 25 accessions belonging to 17 species) were studied using the nuclear ribosomal DNA internal transcribed spacer (ITS) region. The analysis included commercially important species such as melon (C. melo L.) and cucumber (C. sativus). Two additional cucurbit species, watermelon and zucchini, were also included as outgroups. The data obtained reflected the clustering of Cucumis species in four main groups, comprising accessions from cucumber, melon, C. metuliferus and the wild African species. Some of the species clustered in different positions from those reported in classifications previously described by other authors. The data obtained clearly identify a division between the 2n=2x = 14 species (C. sativus) and the 2n = 2x = 24 ones (C. melo and wild species). Within the wild species we identified a subgroup that included C. sagittatus and C. globosus. Oreosyce africana, also classified as Cucumis membranifolius, was shown to be nested within Cucumis. Three accessions previously classified as independent species were shown to be genotypes of Cucumis melo. A set of melon and cucumber SSRs were also used to analyse the Cucumis species and the results were compared with the ITS data. The differential amplification of the SSRs among the accessions made it possible to distinguish three main groups: melon, cucumber and the wild species, though with less detail than applying ITS. Some SSRs were shown to be specific for melon, but other SSRs were useful for producing PCR fragments in all species of the genus.We are grateful to NCRPIS, IPK in Gatersleben, Semillas Fitó S.A., Michel Pitrat and Fernando Nuez for providing seeds. We would also like to thank Vanessa Alfaro, Trinidad Martínez and Núria Galofré for their excellent technical assistance. This work was financed by project AGL2000-0360 of Spains Ministerio de Ciencia y Tecnología (MCYT). AJMs work was supported by a postdoctoral contract from Spains MCYT.     

Primary structures of alpha- and beta-subunits of alpha-amylase inhibitors from seeds of three cultivars of Phaseolus beans

The primary structures of three -amylase inhibitors (TAI, DAI, and MAI-2) consisting of glycoprotein subunits and from the respective seeds of three cultivars of Phaseolus beans, Toramame (Phaseolus vulgaris L.), Daifukumame (Phaseolus vulgaris L.), and Murasakihanamame (Phaseolus coccineus L.) were determined by sequencing the peptide fragments derived from their enzymatic digestions. Major sugar chains of the inhibitors were also assessed by analyzing glycopeptides in the enzymatic digests. The subunits, and , were shown to be composed of 76 and 139 amino acid residues, respectively, in each inhibitor. The overall amino acid sequences of the inhibitors were slightly different from one another. Furthermore, the sequence of TAI was the same as that deduced from a cDNA clone encording -amylase inhibitor-1 from the common bean (Phaseolus vulgaris L.). It was also revealed that there were two N-glycosylation sites in each -subunit: PA-derivatives of the major N-glycans were estimated to be M6B at Asn(12) and M9A at Asn(65). Each -subunit of TAI and MAI-2 had two N-glycosylation sites, while the -subunit of DAI had only one site. The major N-glycans pyridylaminated were estimated to be M3X at Asn(63) in each -subunit and M3FX at Asn(83) in -subunits of TAI and MAI-2.     

Immunosystematical comparison ofPhaseolus coccineus seed proteins with those of other legumes

An immunosystematical research was conducted on the seed proteins of 32 species ofFabaceae. By comparing all immunelectrophoretic patterns with the self reaction of a reference system (Phaseolus coccineus) all proteins detected have been identified and their distribution within the family has been analyzed. Half of the proteins identified inP. coccineus gave positive cross reactions with proteins present in all other species. Among this group are protein I and phaseolin. This result supports the homologization between phaseolin and vicilin. Among the other proteins, three are irregularly distributed throughout the family, and only three are restricted to a few taxa. This last group includes phytohaemagglutinin, which does not present any cross reaction outside the tribePhaseoleae.     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

Steroid Polyols from the Far Eastern Starfish Henricia sanguinolenta and H. leviuscula leviuscula

Two new asterosaponins, (20R)-3-O--D-(2-O-methylxylopyranosyl)-24-propylcholest-4-ene-3,6,8,15,16,29-hexaol (sanguinoside A) and (20R,24S)-3-O--D-(2,3,4-tri-O-methylxylopyranosyl)-5-cholestane-3,4,6,8,15,24-hexaol (sanguinoside B), were isolated from two species of Pacific Far Eastern Starfish Henricia sanguinolenta and H. leviuscula leviuscula, collected in the Sea of Okhotsk. Both glycosides contain aglycones with pentahydroxysteroid nuclei of similar structures, which are substituted at the 3-hydroxy group with differently methylated -D-xylosyl residues. Sanguinoside A has an unusual structure of its aglycone side chain, whereas sanguinoside B has a unique permethylated carbohydrate chain. In addition, laevisculoside G, a known glycoside, was identified in the H. leviuscula starfish. The structures of the isolated glycosides were established by interpreting their spectral data and by comparing their spectral characteristics with those of known compounds.     

Detection and partial characterization of two antigenically distinct β-amylases in developing kernels of wheat

A -amylase (EC 3.2.1.2) was identified in the outer pericarp (P) of developing seeds of wheat (Triticum aestivum L.) and compared with the well known -amylase which is synthesized during seed development in the starchy endosperm (E). The enzyme P already exists in the tissues before anthesis and vanishes at the time when E starts to accumulate. The isoelectric-focusing patterns of P and E are very similar. The relative molecular weight (M_r) of P is slightly higher than that of E (66 and 64.5 kDa, respectively). Both P and E exhibit common epitopes in addition to epitopes specific for each of them. The two enzymes were identified in small amounts in the green tissues of the developing seeds (inner pericarp and testa). No antigenic difference was detected between P and the -amylases of roots and leaves.Abbreviations P pericarp -amylase - E endosperm -amylase - IS1 anti--amylase immune serum - IS2 anti- and anti- amylase immune serum - IS3 anti- amylase immune serum - IEF isoelectric focusing - IgG immunoglobulin G The authors thank Dr. P. Ziegler (Universität Bayreuth, FRG) for stimulating discussion and for useful suggestions during the writing of the text. The authors thank Miss C. Mayer for her skillful technical assistance.     

Ibf-1 (Iodine binding factor), a highly variable marker system in the Triticeae

Summary Isoelectric focusing of extracts from the endosperm of mature grains of hexaploid wheat and related species was used to study the genetic control of Iodine binding factor (IBF). Ten IBF bands were present in Chinese Spring (CS) and analysis of the nullisomictetrasomic and ditelosomic lines of CS showed nine of them to be controlled by genes on the long arms of the homoeologous group 5 chromosomes. Five alleles were detected at Ibf-A1 locus, four at Ibf-B1 and four at Ibf-D1 among a sample of 46 wheat genotypes. Homoeoloci were found on chromosome 5R of Secale cereale, 5E of Agropyron elongatum, 5U of Aegilops umbellulata, 5Agⁱ of Agropyron intermedium, 5S¹ and 4S¹ of Aegilops sharonensis and 4H of Hordeum vulgare.     

Effect of genotype,explant and medium onin vitro regeneration of red pepper

In vitro plant regeneration was achieved inCapsicum praetermissum, C. baccatum andC. annuum cvs. G4, Bhiwapuri Sweet pepper, Cayenne pepper and Hybrid pepper. Shoots were induced from hypocotyl, cotyledon and leaf explants on Murashige and Skoog medium supplemented with 5.7 M indoleacetic acid (IAA)+13.3 M benzyladenine (BA); 22 M BA; and 44 M BA. Analysis of variance revealed that the most significant effect on shoot regeneration was due to the explant and it accounted for 56.3% of total variation observed. The genotype x explant effect on regeneration was minor relative to all other 2- and 3-way interactions because leaf explants consistently regenerated more shoots than hypocotyls or cotyledons in all the genotypes and thereby reduced the variation among the genotypes. Explant x medium interaction revealed that 22 M BA was the best growth regulator supplement in regeneration medium for optimal shoot regeneration from leaf explants. Rooting of regenerated shoots was achieved on 5.7 M IAA-containing medium, and the rooting response was better from shoots induced on medium fortified with 5.7 M IAA plus 13.3 M BA. Complete plantlets with diploid chromosome number (2n=2x=24) were transferred to soil and 60–70% of these plantlets survived and grew well.     

Requirements for in vitro propagation of seven nitrogen-fixing Alnus species

Studies on the in vitro propagation of Alnus crispa, A. glutinosa, A. incana, A. japonica, A. rubra, A. sinuata and A. viridis indicated interspecific as well as intraspecific variations in their requirements for in vitro culture. The WPM and Blaydes media supported, respectively, growth of A. glutinosa and A. crispa but not that of both species, while the MS medium induced equal or significantly better growth than WPM and Blaydes media for both species. The optimum type and concentration of sugar to be used in the multiplication medium varied with species. Only A. glutinosa showed good growth on sucrose while glucose was optimum for all other species but at different concentrations. All species rooted in 3 weeks on half-strength MS medium including 1 M IBA. All clones of A. glutinosa and A. rubra rooted 100%, whereas easy-to-root and difficult-to-root clones were observed in the other species. In the rooting medium, glucose promoted rooting of the difficult-to-root clones better than sucrose. Survival following transfer to an artificial substrate was 100% for all species. Nodulation tests using pure cultures of two Frankia strains showed 100% nodulation on all Alnus clones.     

N-andO-alkylation of glycoconjugates and polysaccharides by solid base in dimethyl sulphoxide/alkyl lodide

O-Methylation of simple neutral oligosaccharides is readily accomplished in dimethyl sulphoxide containing solid sodium hydroxide and methyl iodide [Cincanu I, Kerek F (1984) Carbohydr Res 131209-17]. This procedure has been extended to 2-acetamido-2-deoxy sugars and sialic acid-containing oligosaccharides. CompleteO-andN-methylation was in most cases achieved in 15 min. Esterification of carboxylic groups in uronic acids was fast and resulted in concomitant -elimination. The method is also suitable for methylation of glycoproteins and glycosphingolipids. Polysaccharides can also be methylated by this technique. Analysis of the products by gas-liquid chromatography and mass spectrometry showed no degradation products.Abbreviations lacto-N-tetraose LcOse₄, Gal3GlcNAc3Gal4Glc - lacto-N-fucopentaose III III³Fuc-nLcOse₄, Gal4[Fuc3]GlcNAc3Gal4Glc - trihexosylceramide GbOse₃Cer, Gal4Gal4Glc1-1Cer - globoside GbOse₄Cer, GalNAc3Gal4Glc1-1Cer - FAB-MS fas atom bombardment mass spectrometry     

The temperature-dependent duration of development and parasitism of three cereal aphid parasitoids, Aphidius ervi, A. rhopalosiphi, and Praon volucre

Temperature dependencies were established for the egg-to-mummy and mummy-to-adult phases, for mummy mortality, and for parasitism of Aphidius ervi Haliday, Aphidius rhopalosiphi De Stefani-Perez, and Praon volucre (Haliday) (Hymenoptera, Aphidiidae), three parasitoids of Sitobion avenae (Fabricius) (Homoptera, Aphididae), at 8°C, 12°C, 16°C, 20°C, and 25°C on winter wheat (cv. Haven). A physiological model described temperature-dependent development over the full temperature range, whereas a linear model was fitted for data above 8°C and used to estimate the lower temperature thresholds and day-degrees (° D) required for development. The thresholds for A. ervi were 2.2°C for egg-mummy development and 6.6°C for mummy-adult development, those for A. rhopalosiphi were 4.5°C and 7.2°C, and those for P. volucre were 3.8°C and 5.5°C. The time to develop into mummies and adults differed significantly between the three species: A. ervi development into mummies required an average of 159 ° D, while development into adults took an average of 73 ° D. The corresponding average times required for A. rhopalosiphi and P. volucre to develop mummies were 124° D and 126° D, while their development into adults required an average of 70° D and 150° D, respectively. Mummy mortality was 25–35% at 8°C and less at the higher temperatures tested, but began to increase again at 25°C, showing a quadratic relationship between mortality and temperature. Parasitization was very low or, in the case of P. volucre, absent up to 12°C and thereafter increased with increasing temperature. The relationship between parasitization, recorded as percent aphids mummified, and temperature was linear at the temperatures tested and depended on species. A. ervisuperparasitized 11.1% aphids at 20°C and 16.6% aphids at 25°C, whereas superparasitism was low in A. rhopalosiphi and absent in P. volucre. From 16°C to 25°C the P. volucre sex ratio increased. For A. ervi and A. rhopalosiphi there was no trend with temperature, but at 20°C and 25°C it was close to even. Field data for 1996 and 1997 allowed for a comparison of actual and expected emergence of overwintering mummies. In both years, parasitoids were predicted to have emerged from overwintering mummies well in advance of the onset of aphid infestation, and more than a month earlier than the first parasitized aphids were found in winter wheat. Observations from trap plants in other crops supported the predictions of the models. Other factors that can affect biological control by cereal aphid parasitoids are discussed.     

Intraspecific classification of melons (Cucumis melo L.) in view of their phenotypic and molecular variation

Cucumis melo L. (melon) genotypes differ widely in morphological and biochemical traits. Intraspecific classification of such variability has been difficult, and most taxonomists still rely on the work of Naudin (1859). A collection of 54 accessions representing diverse genotypes from 23 countries was surveyed. Morphological traits related to the vegetative and flowering stages and mature fruit morphology and quality parameters, e.g., taste, aroma, sugar composition and pH, were scored. These were used to construct a botanical-morphological dendrogram that generally reflected the classification ofCucumis melo into several horticultural varieties. DNA polymorphism among the accessions was assessed using the Inter-SSR-PCR and RAPD techniques that detected abundant DNA polymorphism among melon genotypes. Cluster analysis indicated that the largest divergence was between North American and Europeancantalupensis andinodorus cultivars as one group, and the more exotic varieties:conomon, chito, dudaim, agrestis andmomordica, as a second group. The molecular phylogeny agreed, broadly, with the classification of melon into two subspecies, and did not contradict the division into horticultural varieties. It was apparent, however, that the infra-specific division is rather loose, molecular variation being distributed continuously between and within cultivar groups. We suggest that despite the morphological diversity, separation between varietal-groups may be based on a too small number of genes to enable unambiguous infra-specific classification based on DNA diversity.     

Contribution to the knowledge of the enzymatic activity of yeasts of clinical interest

The determination of the enzymatic activity of the yeasts has been applied to the identification of species, specially that ofCandida albicans. In order to know its usefulness in species of clinical interest, we have tested the commercial system API ZYM (Bio Mérieux) on 500 isolated strains of different organic samples, belonging to eight genera and twenty species. All the strains showed positivity to Phosphatase alcaline, Esterase (C4), Esterase lipase (C8), Leucine arylamidase and Phosphatase acid, and negativity to Lipase (C14), Trypsin, Chymotrypsin, -galactosidase, -glucoronidase, -manosidase and -fucosidase. Fourteen enzymatic activity patterns were obtained considering the substrates with variable results for the whole of the strains: Valine arylamidase, Cystine arylamidase, Naphthol-AS-BI-phosphohydrolase, -galactosidase, -glucosidase, -glucosidase and N-acetyl--glucosaminidase. In the majority of the species, the enzymatic profile did not have very specific results since it is usually shared by more than one species.C. albicans is that which presents the greatest number of enzymatic variations, some of these are similar to those of other common clinical species, such asCandida krusei, Candida parapsilosis andCandida tropicalis. This system is proposed as a rapid method for identification and as an epidemiological marker of medically important yeasts.Abbreviations AGL -glucosidase - BGA -galactosidase - BGL -glucosidase - CAA Cystine arylamidase - NAG N.Acetyl--glucosaminidase - PHO Naphthol-AS-BI-phosphohydrolase - VAA Valine arylamidase     

Sex determination in Sarotherodon (tilapia)

Summary The simplest possible model of the sex determination process adding autosomal influence to a minimal number of sex chromosomes was developed to explain matings of Tilapia (Sarotherodon) species. Eighteen different genotypes, each having two autosomes (AA, Aa, or aa) and two sex chromosomes (WX, WY, WW, XY, XX or YY) involved in sex determination, are predicted by the theory. Their sex (10 males and 8 females) were determined using a series of directed graphs, showing the relative strength of the chromosome pairs, developed on the basis of Chen's sex ratio results (Chen 1969). This theoretical model predicts eight different sex ratios (01, 13, 35, 11, 97, 53, 31, 10 ); three of them are not predicted by the WXYZ theory. The greatest part of these sex ratios have been obtained experimentally in extensive series of crosses between related species of Tilapia and their hybrids, carried out by several authors. The theory succeeds in explaining all of Chen's results, including those ratios 53 and 01 seen in certain crosses but not predicted by the WXYZ theory. The importance of the autosomes is seen in comparisons of the genotype pairs (AaWY, aaWY), (AaXY, aaXY) and (AAWW, AaWW) in which the first genotype in each case is male while the second is female as proven by the sex ratio results. The members of the pair differ only in the substitution of one autosome for the other. To test the theory, experiments consisting of hormonal sex reversion and a series of crosses are proposed. Finally, theoretical and practical implications of the theory are discussed.     

Simplified typing of mouse hemoglobin (Hbb) phenotypes using cystamine

Cellulose acetate electrophoresis of mouse hemoglobins modified with the disulfide reagent cystamine permits rapid, unequivocal discrimination of all combinations of the codominant mouse hemoglobin single (Hbb ^s ) and diffuse (Hbb ^d and Hbb ^p ) alleles. The single, diffuse major, diffuse d-minor, and diffuse p-minor adult hemoglobins are all resolved by this method, which depends on the presence of a cysteine in the chains of diffuse mice which is not found in the chain of single mice.This work was supported by research grants ACS-VC58 and NIH CA-01074. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Salmonella typhimurium LT2 metF operator mutations

Summary Using an Escherichia coli lac deletion strain lysogenized with lambda phage carrying a metF-lacZ gene fusion (Flac), in which -galactosidase levels are dependent on metF gene expression, cis-acting mutations were isolated that affect regulation of the Salmonella typhimurium metF gene. The mutations were located in a region previously defined as the metF operator by its similarity to the E. coli metF operator sequence. Regulation of the metF gene was examined by measuring -galactosidase levels in E. coli strains lysogenized with the wild-type Flac phage and mutant Flac phage. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product, but not the vitamin B₁₂ control system mediated by the metH gene product. The results also demonstrate that negative control of the metF gene by the metH gene product and vitamin B₁₂ is dependent on a functional metJ gene product.Abbreviations Ap ampicillin - dNTP deoxyribonucleoside triphosphates - GM glucose minimal - Km kanamycin - L-agar Luria agar - LM lactose minimal - SAM s-adenosyl-L-methionine - TPEG phenylethyl -D-thiogalactoside - X-gal 5-bromo-4-chloro-3-indolyl -D-galactopyranoside - [] designates plasmid-carrier state - :: novel joint     

Copper but not iron limitation increases astaxanthin production by Phaffia rhodozyma in a chemically defined medium

The influence of copper (0–32 M) and iron (0–108 M) on growth and astaxanthin production by Phaffia rhodozyma was studied. Copper below 3.2 M increased the astaxanthin content of the cells (from 220 to 287 g g^–1) but at the expense of a slightly decreased growth (from 11.3 to 10.2 mg ml^–1). In contrast, iron below 1 M decreased both the growth and astaxanthin content of the cells. Using copper limitation instead of toxic respiratory inhibitors to improve astaxanthin production has obvious advantages from the product quality, environmental and process operation points of view.     

Proliferation and germination of somatic embryos from embryogenic suspension cultures in <Emphasis Type="Italic">Phœnix dactylifera</Emphasis>

The present study demonstrates a procedure for the rapid development of a high number of somatic embryos from embryogenic suspension culture. This method might be efficient for mass propagation of Phnix dactylifera L. Embryogenic callus placed in liquid medium with 10^–5M ABA yielded an average 72 embryos per 100ml of culture medium within 2months, while those placed on solid medium yielded an average of 33, 20 and 16 embryos per 100ml of culture medium respectively for 10^–7, 10^–6 and 10^–5 M ABA after 4months. The combination of 2,4-DIchlorophenoxyacetic acid (2,4-D) (4.5×10^–7M), glutamine (6.7×10^–4M), and ABA (10^–5M) (L8 liquid medium) showed a beneficial effect on somatic embryos production compared to 2,4-D and glutamine alone, while this combination significantly (p<0.05) increased the accumulation of storage proteins (144 and 138mgg^–1 DW respectively for Jihel and Bousthami noir cultivars) in somatic embryos. The somatic embryos which underwent maturation on medium containing only 4.5×10^–7M 2,4-D and 10^–5M ABA (L6 liquid medium) accumulated more sugars (292 and 265mgg^–1 DW respectively for Jihel and Bousthami noir) than those matured on any other liquid medium. Histological studies revealed that somatic embryos (developed in L6 and L8 liquid media) accumulated less reserve compounds (proteins and sugars) than zygotic embryos. The addition of activated charcoal (0.25 and 0.5gl^–1) and phytagel^® (2.5gl^–1) to the germination medium may be useful for enhancing the germination of Phnix dactyliferasomatic embryos.