COMPARISON OF PLANT DNA CONTENTS DETERMINED BY FEULGEN MICROSPECTROPHOTOMETRY AND LASER FLOW CYTOMETRY

An improved procedure is reported for determining DNA amounts of plant nuclei. Nuclei stained with propidium iodide, isolated from chopped plant leaves, were passed through an Ortho Cytofluorograph with a Lexel model 95 argon laser (514 nm) and the fluorescence measured, integrated, and recorded using an Ortho 2140 Data Acquisition computer. All nuclear samples were mixed with nuclei of Sultan barley (2C DNA content = 11.12 pg [picogram]) as an internal standard. DNA contents of ten plant species, ranging from 2C = 1.7 pg to 36.1 pg measured by flow cytometry, correlated strongly (r = 0.99, slope = + 1.00) with DNA contents determined from Feulgen-stained nuclei of the same species using microspectrophotometry. The flow cytometric procedures were sufficiently sensitive to detect differences in DNA content between inbred lines of corn and their F1 hybrids. Our results obtained with improved procedures, specifically using propidium iodide as a fluorochrome and plant nuclei instead of chicken erythrocytes as an internal standard, demonstrate that laser flow cytometry can be a precise, rapid, and reliable method for determining nuclear DNA content of plants.     

POPULATION GENETIC STRUCTURE IN DIPLOID BLUEBERRY VACCINIUM SECTION CYANOCOCCUS (ERICACEAE)

Vaccinium section Cyanococcus comprises diploid, tetraploid, and hexaploid species (x = 12) all of which are highly self-sterile. In order to assess the distribution of genetic variation within and among the diploids, population genetic analyses of allozyme data were conducted on three species: V. elliottii, V. myrtilloides, and V. tenellum. Populations were located throughout the range of these taxa in eastern North America. Data were collected at 20 loci, 12 of which are mendelian based on formal genetic analyses. Consistent with expectations for out-crossing taxa, these species exhibited high levels of variation within populations. Eighteen loci were polymorphic with the number and frequency of alleles varying among taxa. Mean values for the number of alleles per polymorphic locus, proportion of polymorphic loci, and expected heterozygosity were 2.8, 48.2, and 0.148, respectively. High infraspecific genetic identities exceeding 0.9 indicated that these taxa are homogeneous. All populations were in Hardy-Weinberg equilibrium with slight heterozygote excess observed in V. myrtilloides and V. tenellum. Although total genetic diversity was lower than that observed for other predominantly out-crossing species, it was apportioned similarly. The majority could be attributed to differences among individuals within populations. Conspecific populations were relatively undifferentiated with genetic differentiation similar to other self-incompatible species.     

Internode length in Pisum. Estimation of GA1 levels in genotypes Le, le and led

The levels of GA₁, 3-epiGA₁ and GA₈ in genotypes Le, le and le^d of Pisum sativum L. were determined by gas chromatography-selected ion monitoring (GC-SIM) after feeds of [³H, ¹³C]-GA₂₀ to each genotype. The levels of endogenous and [¹³C]-labelled metabolites were determined by reverse isotope dilution with unlabelled GA₁, 3-epiGA₁ and GA₈. The results demonstrate a quantitative relationship between the level of GA₁ and the extent of elongation both on a per plant and a per g fresh weight basis. These results are consistent with previous findings in peas and other species possessing a predominant early 13-hydroxylation pathway for GA biosynthesis.
The levels of 3-epiGA₁ also decreased in the genotypic sequence Le, le, le^d although not as rapidly as for the level of GA₁. This may suggest that the alleles at the le locus also influence the formation of 3-epiGA₁.     

INFLUENCE OF CELL SHAPE AND SIZE ON ALGAL COMPETITIVE ABILITY1

Allometric relations between physiological processes and cell volume and surface area are combined with the variable-internal-stores model of growth to predict the ability of hypothetical phytoplankton to compete for phosphorus at equilibrium. The analysis shows that for spherical cells, smaller cells are better competitors than large ones. For cells that are very elongated in shape, however, large cells are often better competitors than small ones. The cells predicted to be the best competitors compare favorably in size and shape with the species observed to dominate in phosphorus-limited chemostats at equilibrium.     

EVIDENCE FOR POST-TRANSLATIONAL MODIFICATION OF TRIOSE PHOSPHATE ISOMERASE (TPI) IN ISOËTES (ISOËTACEAE)

As part of an electrophoretic study on Isoëtes, a number of Neotropical and North American species were examined for allozyme variation in TPI. Three of these species—I. storkii, I. flaccida, and I. mexicana—exhibit three distinct zones of TPI activity. The two most anodally migrating zones are comparable to the two zones found in most angiosperms and in several other species of Isoëtes. The single or three-banded phenotypes produced at these loci correspond, respectively, to the homozygous and heterozygous patterns typical of a dimeric enzyme. The most cathodal zone (zone III) differs in producing either single or two-banded phenotypes. Analyses of these three zones indicate a nearly perfect correlation between zones II and III in putative allelic constitution and relative allelic mobility. Explanations involving TPI gene duplications and/or null alleles fail to account for the peculiar banding characteristics and origin of activity zone III. An alternative hypothesis involving a protease duplication and differential post-translational modification is postulated. This hypothesis adequately explains the zone III phenotypes and fixation of the third activity zone in the species examined. Amino acid sequencing is suggested as the most direct test of this hypothesis. The taxonomic distribution of TPI III generally supports a previous, morphologically-based, hypothesis on species relationships in Isoëtes. The presence of this zone is regarded as an independent synapomorphy for a major clade of Neotropical Isoëtes.     

Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional 1H homonuclear and 13C-1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.     

A SURVEY OF SIZES AND WEIGHTS OF BEMISIA TABACI (HOMOPTERA:ALEYRODIDAE)B BIOTYPE LIFE STAGES FROM FIELD GROWN COTTON AND CANTALOUPES

Abstract Size and weight measurements were made for all the life stages of Bemisia tabaci (Gennadius) B biotype from field grown cotton ( Gossypium hirsutum L.) and cantaloupe ( Cucumis melo L., var. cantalupensis ) in Phoenix, AZ and Fargo, ND, USA in 2000 and 2001. Nymphal volumes were derived from the measurements. The average nymphal volume increase for settled 1 st to the late 4th instar was exponential. The greatest increase in body volume occurred during development from the 3rd to early 4th instar. Nymphs on cotton leaves were wider, but not longer compared with those on cantaloupe. Ventral and dorsal depth ratios of nymphal bodies from 1 st to late 4th instars from cantaloupe leaves were significantly greater compared with those from cotton leaves. During nymphal development from 1st to 4th instar, the average (from the two host species) ventral body half volume increased by nearly 51 times compared with an increase of 28 times for the dorsal body half volume. Adult female and male average lengths, from heads to wing tips, were 1 126 μm and 953 μm, respectively. Average adult female and male weights were 39 and 17 μg, respectively. Average widths, lengths, and weights of eggs from cotton and cantaloupe were, 99 μm, 197 μm, and 0.8 μg, respectively. Average widths, lengths, and weights for exuviae of non-parasitized nymphs from both cotton and cantaloupe were 492 μm, 673 μm, and 1.20 μg, respectively; and widths, lengths, and weights of parasitized nymph exuviae were 452 μm, 665 μm, and 3.62 μg, respectively. Both exuviae from non-parasitized and parasitized nymphs from cotton leaves were wider, longer, and heavier than those from cantaloupe leaves.     

Single embryo transfer in IVF to prevent multiple pregnancies.

As the success rates of IVF clinics improve, one of the adverse consequences is the increased incidence of twins, due largely to the number of embryos transferred. Even if the number of embryos transferred is restricted to two, the twinning rate can exceed 40% of the pregnancies. An obvious way to reduce this high twin rate would be to transfer only one embryo. This would require that cryopreservation of the supernumerary embryos be efficacious enough so that the chance of achieving an ongoing pregnancy is not diminished by transferring a single embryo in the stimulated cycle. Previous studies utilising embryos on day 2 and 3 of development have shown that the pregnancy rates can be acceptable (about 40%) and that the cumulative rate can be up to 60%. Most of these studies, however, do not include a comparison with the cumulative pregnancy rate with two embryos transferred in the stimulated cycle. Therefore, the efficacy has not been proven. We present clinical data from the past few years to illustrate the increase in success rates and the concomitant increase in twinning rates. The increased success in the cryopreservation program has enabled us to trial a single embryo transfer program and compare the results to the transfer of two embryos. The results strongly suggest that the transfer of a single embryo is the better clinical option.