Xenopus nucleoplasmin: egg vs. oocyte

Nucleoplasmin has been purified from either oocytes or unfertilized eggs of the frog, Xenopus laevis. We find that the pentameric form of egg nucleoplasmin exhibits an apparent molecular mass approximately 15 000 daltons larger than its oocyte counterpart upon sodium dodecyl sulfate (SDS)-acrylamide gel electrophoresis. Egg nucleoplasmin monomers are more heterogeneous, substantially more acidic, and overall larger in apparent molecular weight than oocyte nucleoplasmin monomers when analyzed by isoelectric focusing or SDS gel electrophoresis. Protease digestions indicate that the structural differences between egg and oocyte nucleoplasmin are primarily confined to the N-terminal halves of the proteins. The structural diversity observed is accompanied by a difference in the ability of nucleoplasmin from the two sources to act as a nucleosome assembly agent in vitro. Egg nucleoplasmin efficiently promotes the formation of nucleosomes onto circular pBR322 DNA in vitro at physiological ionic strength and at physiological histone:DNA ratios, while oocyte nucleoplasmin is markedly deficient in serving as an in vitro chromatin assembly agent under all conditions which we have tested. Treatment of egg nucleoplasmin in vitro with alkaline phosphatase demonstrates that the structural diversity between egg and oocyte nucleoplasmin results primarily from extensive additional phosphorylation of the egg protein. The relevance of nucleoplasmin phosphorylation in leading to differences in the chromatin assembly activity of this protein both in vitro and in vivo is considered.     

Mass fractionation of Drosophila egg chambers.

Egg chambers from Drosophila ovaries may now be fractionated in large quantities suitable for biochemical work. A suspension of isolated egg chambers is prepared by subjecting ovaries to digestion with 0.067% collagenase. Egg chambers are separated in size classes (which correspond to different developmental stages) by sieving them through a column of superimposed nylon nets. The method can, in principle, be used for the separation of any particles ranging upward from 10 μm in diameter.     

Magnetic anisotropy of egg lecithin membranes.

Magnetic realignment and rotational diffusion of cylindrical egg lecithin vesicles were measured under a phase contrast microscope. The anisotropy of magnetic susceptibility times membrane thickness was calculated from the data for several thin-walled vesicles. The resulting values were assigned to discrete numbers of bilayers. The difference between the susceptibilities parallel and perpendicular to the long axes of the lecithin molecules is deduced to be X parallel - X perpendicular = -(0.28 +/- 0.02) . 10(-8) cgs at 23 degrees C, if a bilayer thickness of 60 A is assumed.     

Studies on spin-labelled egg lecithin dispersions.

ESR spectra of egg lecithin dispersions labelled with 5-nitroxide stearic acid are recorded with a 50 G field sweep, and also with a new technique which "expands" the spectrum by (1) recording pairs of adjoining peaks with a smaller field sweep and (2) superposing the common peaks. The expansion technique improves the precision of the order parameters determined from the hyperfine splitting measurements, and may prove useful in future spin label membrane studies. Approximate order parameters are derived to describe the fluidity of fatty acid spin-labelled membranes in those cases where either the inner or outer hyperfine extrema are not well defined. The ability of these expressions to measure the fluidity of labelled egg lecithin disperions for the temperature range 14-42 degrees C is examined.     

Chemical nature of the egg shell in helminths. II. Mode of stabilization of egg shells of monogenetic trematodes

The chemical nature of the egg shells of the monogenetic trematodes Pseudomicrocotyle sp. and Pricea multae, parasitizing the gills of the marine fish Scomberomorus guttatus was studied with a view to understanding the mode of stabilization of the egg shells.The egg shells of the monogenetic trematodes appeared to contain dimers of tyrosine; a conclusion based on fluorescence properties, affinity to toluidine blue, methylene blue, and a positive test for aromatic amino acids. In addition, they are stabilized by -S-S- linkages as indicated by the positive reaction to tests for disulfides. This is also suggested by the shells being refractile to stain but which can be reversed after treatment with sodium thioglycollate.Absence of phenolic tanning in the egg shells of the monogenetic trematodes is indicated by the negative reaction to tests for quinones, the failure to develop color on incubation in catechol after heat treatment, and by the fluorescence in uv light.In these respects the egg shells of the monogenetic trematodes strongly resemble the egg shell of Fasciola hepatica.     

Oligonucleotide site-directed mutagenesis in Xenopus egg extracts.

Addition of M13mp18 single-stranded DNA annealed with an oligonucleotide to a Xenopus egg extract results in a rapid and efficient incorporation of the oligonucleotide in a complete double-stranded supercoiled molecule. Both the efficiency of DNA synthesis and the recovery of complete double-stranded molecules are increased relative to the reaction carried out by the classical technique using the E. coli Klenow DNA polymerase, DNA ligase, dNTPs, ATP and ions. Site specific mutagenesis was assayed by reverting a point mutation in the lacz region of M13mp18. The color assay described by Messing and sequencing of the DNA extracted from isolated plaques was used to check for the reversion. A 2 hr incubation of the heteroduplex carrying the mutagenic oligonucleotide in the Klenow-ligase-dNTP mixture allows a recovery of 6% mutant phage after transformation of competent cells with the reaction products. Using the Xenopus egg extract, 83% mutant phage were recovered after the same incubation time, in reactions entirely performed in parallel. The Xenopus extract is stable and contains all components required for the assay, including all ionic and protein factors; thus the only addition is the annealed DNA. Such an eukaryotic system is therefore an attractive alternative to the reconstituted prokaryotic DNA polymerase-DNA ligase system for site specific mutagenesis.     

Activity of crystalline turkey egg white lysozyme.

Hexagonal crystals of turkey egg white lysozyme have been examined for activity in order to evaluate their potential for use in time-resolved X-ray crystallographic experiments. Substrates used in this study were hexa-N-acetylglucosamine (hexa-GlcNAc) and a modified analogue of hexa-GlcNAc where the terminal sugar ring was opened by reduction with tritiated sodium borohydride. This gave a labeled beta-N-acetylglucosaminitol unit at the sixth position of the sugar chain and allowed easy quantitation of enzymatic cleavage on TLC plates. Using these substrates, it has been shown that turkey egg white lysozyme is enzymatically active in the crystal. Enzyme dispersed in the buffer surrounding the crystal does not show detectable activity under conditions relevant to an X-ray experiment. Unmodified hexa-GlcNAc is hydrolyzed into di-, tri-, and tetrasaccharides in the crystal. This cleavage pattern is different from that obtained with hen egg white lysozyme in solution and likely causes of the differences are discussed. The reduced radiolabeled oligosaccharide has a unique cleavage pattern with trisaccharides as the products. The specific activity of the enzyme with the radiolabelled analogue was 9.8 (+/- 1.0) x 10(-7) mmol/min/mg protein at 22 degrees C in the crystal.     

Occurrence of glycosphingolipids in chicken egg yolk.

Chicken egg yolk was found to contain a unique glycosphingolipid pattern not seen in other types of tissue or cell. These glycosphingolipids were isolated in pure form and their structures established by sequential enzymic hydrolysis and permethylation analysis. The major gangliosides in chicken egg yolk are N-acetylneuraminosylgalactosylceramide, N-acetylneuraminosyl-lactosylceramide and di-N-acetylneuraminosyl-lactosylceramide. The only neutral glycosphingolipid found in chicken egg yolk is galactosylceramide.     

Exploring levels of egg parasitism and variation in egg cuticular chemistry in different Clavigralla spp.

Clavigralla spp. (Hemiptera: Coreidae) are major pests of cowpea (Vigna unguiculata (L.) Walp, Fabacae), common bean (Phaseolus vulgaris L., Fabacae) and pigeon pea (Cajanus cajan L., Fabacae) in Africa. Clavigralla spp. egg parasitoids, Gryon spp. (Hymenoptera: Scelionidae), have previously been reported as potential biological control candidates. Little is known about the parasitism levels and their potential relationship with cuticular chemistry of Clavigralla spp. The aims of this study were to determine parasitism levels of Clavigralla tomentosicollis Stål (Hemiptera: Coreidae) and C. elongata Signoret (Hemiptera: Coreidae) eggs, and to explore the relationship between egg parasitism and egg cuticular chemistry. High parasitism levels were determined for C. tomentosicollis by collecting eggs from plants in mono‐cropping and multi‐cropping systems in farmers’ fields in Bénin and Kenya between April and June 2016. Three species of Clavigralla were recorded: C. tomentosicollis, C. shadabi and C. elongata. Clavigralla tomentosicollis was the most common in both countries, while C. shadabi and C. elongata were only collected in Bénin and Kenya, respectively. An egg parasitoid (Gryon sp.) was recovered from egg batches collected from both countries. In parasitism assays using Gryon sp., the incidence of parasitism was higher in C. tomentosicollis eggs than that of C. elongata. Chemical analysis by coupled gas chromatography/mass spectrometry (GC/MS) of cuticular extracts obtained from C. tomentosicollis and C. elongata eggs identified fifteen compounds including ten alkanes of which the amounts varied between the two species. We speculate that Clavigralla spp. cuticular chemistry may serve as potential host location cues for Gryon sp.