Characterization of a single glycosylated asparagine site on a glycopeptide using solid-phase Edman degradation

The characterization of site-specific glycosylation is traditionally dependent on the availability of suitable proteolytic cleavage sites between each glycosylated residue, so that peptides containing individual glycosylation sites are recovered. In the case of heavily glycosylated domains such as theO-glycosylated mucins, which have no available protease sites, this approach is not possible. Here we introduce a new method to gain site-specific compositional data on the oligosaccharides attached to a single amino acid. Using a model glycopeptide from a mutant human albumin Casebrook, glycosylated PTH-Asn was recovered after sequential solid-phase Edman degradation, subjected to acid hydrolysis and the sugars were identified by high performance anion exchange chromatography with pulsed amperometric detection. The PTH-Asn(Sac) derivative was further characterized by ionspray mass spectrometry. Comparison between an endoproteinase Glu-C glycopeptide and a tryptic glycopeptide showed that the oligosaccharide attached to Asn494 was stable after at least 10 cycles of Edman degradation.     

Time series canopy phenotyping enables the identification of genetic variants controlling dynamic phenotypes in soybean

Advances in plant phenotyping technologies are dramatically reducing the marginal costs of collecting multiple phenotypic measurements across several time points. Yet, most current approaches and best statistical practices implemented to link genetic and phenotypic variation in plants have been developed in an era of single-time-point data. Here, we used time-series phenotypic data collected with an unmanned aircraft system for a large panel of soybean (Glycine max (L.) Merr.) varieties to identify previously uncharacterized loci. Specifically, we focused on the dissection of canopy coverage (CC) variation from this rich data set. We also inferred the speed of canopy closure, an additional dimension of CC, from the time-series data, as it may represent an important trait for weed control. Genome-wide association studies (GWASs) identified 35 loci exhibiting dynamic associations with CC across developmental stages. The time-series data enabled the identification of 10 known flowering time and plant height quantitative trait loci (QTLs) detected in previous studies of adult plants and the identification of novel QTLs influencing CC. These novel QTLs were disproportionately likely to act earlier in development, which may explain why they were missed in previous single-time-point studies. Moreover, this time-series data set contributed to the high accuracy of the GWASs, which we evaluated by permutation tests, as evidenced by the repeated identification of loci across multiple time points. Two novel loci showed evidence of adaptive selection during domestication, with different genotypes/haplotypes favored in different geographic regions. In summary, the time-series data, with soybean CC as an example, improved the accuracy and statistical power to dissect the genetic basis of traits and offered a promising opportunity for crop breeding with quantitative growth curves.     

A model for the pattern of deposition of microfibrils in the cell wall of Glaucocystis

Freeze-fracturing of Glaucocystis nostochinearum Itzigsohn cells during cell-wall microfibril deposition indicates that unidirectionally polarized microfibril ends are localized in a zone of synthesis covering about 30% of the sarface area of the plasma membrane. Within this zone there are about 6 microfibril ends/m² cell surface. It is proposed that microfibrils are generated by the passage of their tips over the cell surface and that the pattern of microfibril organization at the poles of the cells, in which microfibrils of alternate layers are interconnected at 3 rotation centres, results directly from the pattern of this translation of microfibril tips. In a model of the deposition pattern it is proposed that the zone of synthesis may split into 3 sub-zones as the poles are approached, each sub-zone being responsible for the generation of one rotation centre. It is demonstrated that the microfibrillar component of the entire wall could be generated by the steady translation of the microfibril tips (at which synthesis is presumed to occur) over the cell surface at a rate of 0.25–0.5 m min^-1. Microcinematography indicates that the protoplast rotates during cell-wall deposition, and it is proposed that this rotation may play a role in the generation of the microfibril deposition pattern.     

The natural regulation of Giant tortoise populations on Aldabra Atoll. Reproduction

A two-year reproduction study of three isolated populations of different densities of the Aldabra Giant tortoise ( Geochelone gigantea Schweigger) showed dissimilarity in gonadal parameters; (i) as population density increased age at sexual maturity was delayed in males and females; (ii) although the mean number of mature follicles was similar in all populations, preovulatory follicular atresia was only found in the high density population; (iii) annual variation in preovulatory follicular atresia was dependent on rainfall and food availability; (iv) the number of clutches laid/♀/year decreased with increasing population density as did clutch size; in the high density population (and depending on rainfall) only 30–80% of the mature females bred. The discussion considered the importance of movement in determining survival/reproduction.     

Inhibition of thymidine phosphorylase in vivo provides a rapid method for switching DNA labeling

Summary The interaction between nuclear ploidy and chloroplast DNA content was investigated in the unicellular green alga Chlamydomonas reinhardtii. DNA was extracted from both exponentially growing and synchronized haploid and diploid strains and analysed by CsCl equilibrium density centrifugation in an analytical ultracentrifuge. It was found that the doubling of the nuclear genome in diploids was linked to a doubling of the chloroplast DNA content per cell.     

GTP,as well as ATP,can act as a substrate for the intrinsic protein kinase activity of synaptic plasma membranes

Summary GTP as well as ATP can act as phosphate donor for the intrinsic protein kinase activity of synaptic plasma membranes. There are many similarities between the activities observed with ATP or GTP. Both need a divalent cation, Mg²⁺ being preferred, both are slightly inhibited by Na⁺, and more strongly by K⁺, both are inhibited by theophylline and adenosine. The K_m for GTP (0.13 mM) is similar to that ATP (0.12 mM). There are, however, some differences in properties. When GTP instead of ATP is the phosphate donor the pH optimum is 6.5 instead of 7.4. In addition NH ₄ ⁺ inhibits the transfer of phosphate from GTP but not from ATP. More importantly, cyclic AMP only stimulates the transfer of phosphate from ATP not from GTP. SDS gel electrophoresis reveals that similar membrane proteins are phosphorylated by GTP and ATP in the presence or absence of cyclic AMP. This suggests that there may be two different types of protein kinase in the synaptic plasma membrane which act on similar membrane proteins. One is stimulated by cyclic AMP and is specific to ATP while the other is unaffected by cyclic nucleotides and can use either ATP or GTP as phosphate donor.Deceased     

Light-induced changes in the period of the circadian rhythm of carbon dioxide output in Bryophyllum leaves

Summary The period of the rhythm of carbon dioxide output from leaves of Bryophyllum fedtschenkoi R. Hamet et Perrier at 15°C was shorter in continuous white light than in darkness. The period was monitored in leaves exposed to narrow spectral bands of monochromatic radiation at an incident quantum flux density of 4.7×10^-11 einsteins cm^-2s^-1. Bands centred on 660, 600, 730 and 530 nm significantly shortened the period, the greatest effect being achieved at 660 nm and the smallest at 530 nm; those centred on 760 and 450 nm were without effect. None of the bands tested significantly lengthened the period. The period of the rhythm in leaves exposed continuously to monochromatic radiation at 660 nm decreased with increasing quantum flux density. The extent to which a quantum flux density of 4.7×10^-11 einsteins cm^-2s^-1 at wavelength 660 nm shortened the period depended on the ambient temperature. At 15°C a significant reduction of 4.4 h occurred as compared with the dark control, while at 30°C no significant reduction was observed. The transient (the time from the initiation of the rhythm to the first peak) showed a greater dependence on temperature than did the steady-state period. No such difference could be detected in relation to the intensity or quality of irradiation. The reduction of the transient by the various irradiation treatments was, in general, proportional to the reduction of the period.     

Identification of CpG islands around the DXS178 locus in the region of the X-linked agammaglobulinaemia gene locus in Xq22

The X-linked agammaglobulinaemia (XLA) gene locus has previously been mapped to Xq22. Genetic linkage analysis has shown tight linkage between the disease and the DXS178 locus and that DXS3 and DXS94 are the closest proximal and distal flanking markers, respectively, separated by a genetic distance of 10–12 cM. We attempted to construct a physical map of Xq22 using pulsed field gel electrophoresis (PFGE) and rare-cutting restriction enzymes in order to obtain a finite physical value for the distance between DXS3 and DXS94. However, these attempts were hampered by the large number of rare-cutting restriction enzyme sites around the DXS178 locus, indicative of the presence of CpG rich regions of DNA. We were able to construct a physical map of the sites close to DXS178 that suggests the presence of at least three, and perhaps as many as five, CpG islands. These are arranged on either side of DXS178, extending over about 550kb of genomic DNA. Each of these regions must be considered as being associated with a potential candidate gene sequence for the XLA gene and we have initiated a chromosome walk from DXS178 to the nearest of these islands.