Two distinct yolk lipoprotein complexes from Caenorhabditis elegans.

The four yolk polypeptides of the nematode Caenorhabditis elegans are found in two types of lipoprotein particle: 12 S particles with Mr estimated at 450,000 and 8 S particles with Mr estimated at 250,000. Both types of particle contain approximately 8% phospholipids, 3% triglycerides, and 3% other lipids by mass. All four C. elegans yolk polypeptides can be found in either 12 or 8 S particles, depending upon the conditions of isolation. While the properties of the 12 and 8 S lipoprotein particles are consistent with a dimermonomer relationship, the asymmetric distribution of the yolk polypeptides between 12 and 8 S fractions suggests that at least two different oligomeric lipoprotein complexes are present in C. elegans embryos. In order to clarify the subunit composition of the C. elegans yolk lipoproteins, the patterns of polypeptides retained in immunoaffinity binding procedures by immunoglobulins of different antigenic specificities have been compared. When immunoaffinity binding is performed in the absence of sodium dodecyl sulfate, three C. elegans yolk proteins (yp170A, yp115, and yp88) are retained together by polyclonal immunoglobulins directed against either yp115 or yp88. A monoclonal immunoglobulin also retains these three proteins together. In contrast, a second monoclonal immunoglobulin retains only the fourth yolk protein (yp170B). Aggregate species, evidently reflecting the spontaneous formation of interchain disulfide bonds, indicate that yp170A and yp88 are physically associated, whereas yp170B self-associates in dimers. It is concluded that there are two distinct lipoprotein complexes in C. elegans: the A complex, which consists of yp170A, yp115, and yp88 and is essentially heterodimeric and the B dimer, a simple dimer of yp170B.

Interference with function of a homeobox gene in Xenopus embryos produces malformations of the anterior spinal cord.

XIHbox 1 is expressed in a narrow band across the cervical region of Xenopus embryos. The gene produces two related proteins: "long" and "short" XIHbox 1 homeodomain proteins. Injection of antibodies to the long XIHbox 1 protein into 1-cell embryos caused a phenotype in which the anterior spinal cord was morphologically transformed into a hindbrain-like structure. This alteration was restricted to the region normally expressing long XIHbox 1 protein. Injection of long protein mRNA disrupted segmentation and tissue organization without inhibiting cell proliferation. Injection of short protein mRNA into 1-cell embryos produced spinal cord malformations similar, but not identical, to those caused by the antibodies, suggesting antagonistic roles for long and short XIHbox 1 proteins. We immunostained tadpoles carrying extended hindbrains for N-CAM and consistently found defective organization of spinal nerves over the affected region.

Drosophila basement membrane procollagen IV. I. Protein characterization and distribution.

A collagen was isolated from Drosophila E85, Schneider line 2L and Kc cell cultures. The purified protein was characterized and antibodies were raised against it. Immunofluorescence microscopy locates this material to the regions of basement membranes of Drosophila embryos, larvae, and adults. The molecules are mostly, or entirely, homotrimers of one polypeptide chain linked by interchain disulfide bonds. The partial amino acid sequences of a cyanogen bromide cleavage product of this chain are identical with a part of the virtual translation product of the Drosophila pro alpha 1(IV) nucleotide sequence that is reported in the accompanying paper. This gene is at Drosophila chromosome location 25C and was identified by the high homology of one part of it with the noncollagenous carboxyl terminus (NC1) of vertebrate type IV basement membrane collagens (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). In the electron microscope each molecule appears as a thread with a knob at one end, which contains the carboxyl peptide domains. The variation of flexibility of the thread was mapped along its length. Pulse-chase labeling of cell cultures showed that these molecules associate into disulfide-linked dimers and higher oligomers that can be partly separated by velocity sedimentation and are resolved by sodium dodecyl sulfate-agarose gel electrophoresis. Dimers and higher oligomers formed by overlap of the amino ends of molecules were found. Mild pepsin digestion of Drosophila embryos and larvae solubilized the corresponding disulfide-linked collagen molecules, and Staphylococcus aureus V8 protease peptide maps showed the identity of the collagen derived from animals and from cell cultures. Individual, native molecules have a sedimentation coefficient s20,w = 4.1 S, the dichroic spectrum and amino acid composition of a collagen, and a Tm = 31 degrees C. Positive in situ hybridization with a specific probe for this collagen began 6-8 h after egg laying and showed message in the locations of embryos and larvae which reacted with the antibodies. This included some prominent individual cells in the hemolymph.

The dorsal protein is distributed in a gradient in early Drosophila embryos.

dorsal is one of the maternally active dorsal-ventral polarity genes of Drosophila and is closely related to the vertebrate proto-oncogene c-rel. Genetic experiments suggest that dorsal represents one of the last (if not the last) steps in the maternal pathway involved in establishing dorsal-ventral polarity in the early embryo. Even though the dorsal RNA is uniformly distributed in the embryo, we have found that the dorsal protein is specifically localized in peripheral nuclei of syncytial and cellular blastoderm stage embryos, and it is distributed in a ventral-to-dorsal gradient. These findings suggest possible mechanisms for how the dorsal protein may communicate maternal positional information to the zygotic genome.

Vimentin expression in oocytes, eggs and early embryos of Xenopus laevis.

Immunocytochemical studies using a monoclonal anti-porcine vimentin antibody reveal a well-organized pattern of staining in Xenopus laevis oocytes, eggs and early embryos. The positions of Xenopus vimentin and desmin in two-dimensional (2D) polyacrylamide gels were first established by immunoblotting of muscle Triton extracts with anti-intermediate filament antibodies (anti-IFA), which cross-react with all intermediate filament proteins (IFPs). The anti-porcine vimentin reacts with vimentin and desmin in muscle 2D immunoblots, but only reacts with one polypeptide in oocyte blots in the position predicted for vimentin (Mr 55 x 10(3), pI 5.6). Using an anti-sense probe derived from a Xenopus vimentin genomic clone in RNase protection assays, we show that expression of vimentin begins in previtellogenic oocytes. The level of expression remains constant throughout oogenesis and in unfertilized eggs. These data suggest that vimentin is expressed in oocytes and eggs. Most interestingly, the immunocytochemical results also show that vimentin is present in the germ plasma of oocytes, eggs and early embryos. It is therefore possible that vimentin has an important role in the formation or behaviour of early germ line cells.

Conditional absence of mitosis-specific antigens in a temperature-sensitive embryonic-arrest mutant of Caenorhabditis elegans.

A monoclonal antibody, specific to phosphoproteins in mitotic HeLa cells was found to crossreact with a similar set of proteins in embryos of the nematode, Caenorhabditis elegans. In C. elegans, as in mammalian cells, the highly conserved antigenic epitope is associated with a family of high molecular weight polypeptides. The antigenic reactivity of these multiple proteins also depends on their phosphorylation, since antibody binding is reduced after alkaline phosphatase treatment. The antigens are detected at the centrosomes, and in the nuclear region and surrounding cytoplasm of mitotic cells. The significance of these antigens is emphasized by their absence at restrictive temperature in embryos of the temperature-sensitive embryonic-arrest mutant, emb-29V. Furthermore, temperature shift-down experiments suggest that the emb-29 mutation defines a cell division cycle function that affects an essential activity required for progression into M phase.

[Protein fractions of the ova, embryos and skeletal muscles of the loach affecting the stability and activity of lactate dehydrogenase]. / Belkovye fraktsii iaitsekletok, zarodyshei i skeletnykh myshts v'iuna, vliiaiushchie na stabil'nost' i aktivnost' laktatdegidrogenazy.

When lactate dehydrogenase obtained from Misgurnus muscles and purified to the homogeneous state is incubated for 16 hat 38 degrees C, its activity lowers down to 10% of the initial value. Extracts of egg cells, embryos or skeletal muscles of the mentioned fish species added to the enzyme solution decrease considerably its inactivation. Proteins stabilizing the activity of lactate dehydrogenase are revealed in the supernatant liquid obtained after salting out of the above extracts with 60% sulphate ammonium saturation. These proteins are in fractions with the molecular weight below 45 kDa. Among proteins with the molecular weight 10 kDa there are polypeptides which exert an activation effect on lactate dehydrogenase. This effect is intensified with the presence of insulin.

NMR studies of intracellular sodium ions in amphibian oocytes, ovulated eggs, and early embryos.

23Na NMR, in combination with an anionic paramagnetic shift reagent dysprosium bis(tripolyphosphate), has been used to study intracellular Na+ in Rana oocytes, ovulated eggs, and early cleavage embryos. The technique allows accurate and simultaneous determination of both extracellular space and intracellular Na+ concentration. In prophase-arrested, follicle-enclosed oocytes, only about 17% of the total oocyte Na+ (approximately 40 mmol/kg of cells) was NMR-visible. Homogenizing oocytes in 0.24 M sucrose did not significantly affect the 23Na resonance. About 30% of the total oocyte Na+ was associated with the yolk platelets isolated at room temperature by differential centrifugation. NMR analysis, however, did not yield a detectable 23Na signal from these intact platelets. Thus, while yolk platelets are rich in Na+, this Na+ does not contribute to the oocyte 23Na NMR signal. Denuded oocytes, obtained by removing the follicular epithelium, gained about 10 mmol of total Na+/kg of cells and exhibited a comparable increase in NMR-visible Na+, suggesting the existence of compartments with varying degree of NMR visibility within the oocyte. Partially relaxed 23Na Fourier transform NMR spectra revealed the existence of at least two major intracellular compartments of NMR-visible Na+ with different magnetic environments and relaxation behavior in denuded oocytes. Since platelet Na+ appears to be NMR-invisible, one of the two observed compartments may be the nucleus. Progesterone action on the amphibian oocyte caused measurable changes in NMR-visible Na+. By ovulation (second metaphase), there is a gain in total egg Na+, and the NMR-visible Na+ is also increased. Following fertilization, however, there is some loss of total cell Na+ but, by the 2-4 cell stage, about 70% of the total Na+ becomes NMR-visible. These results indicate that a sizable fraction of the Na+ in follicle-enclosed, prophase oocyte is sequestered and located in NMR-invisible compartments and that changes in NMR-visible intracellular Na+ occur following hormonal and developmental stimuli.

Changes in the nuclear lamina composition during early development of Xenopus laevis.

Changes in protein composition of the nuclear lamina were monitored during early development in Xenopus. Lamin LIII, the only lamin present in oocyte nuclei, serves as a lamin pool for the formation of pronuclei and early cleavage nuclei. It is present in embryos up to the tail bud stages. Lamins LI and LII, the lamins originally found in adult cell nuclei, appear at characteristic times in development. LI first appears at the midblastula transition (MBT), and LII at the gastrula. Tryptic peptide analysis revealed that all three lamin forms found in the embryo are identical with the adult lamins. De novo synthesis of LIII and LI, observed at MBT, is independent of transcription and must therefore be due to activation of maternal mRNAs. These results are discussed in relation to other nuclear changes occurring during early development.

Analysis of the membrane-associated poly(A)+RNA in the cytoplasm of dormant Artemia cysts by DNA excess hybridization. Evidence for a nuclear origin.

Encysted embryos of Artemia contain latent mRNA, to a large extent associated with a fraction of cytoplasmic membranes. The membranes, purified by EDTA treatment and banding in a sucrose gradient at 1.17 g/cm3, include endoplasmic vesicles and mitochondria. The origin of the membrane-associated poly(A)+RNA was therefore investigated. In gel electrophoresis poly(A)+RNA from the purified membranes of dormant cysts forms two distinct bands at approx. 3 . 10(5) and 5 . 10(5) Da. Later during development the lighter component decreases. Nuclei from dormant cysts are devoid of poly(A)+RNA, while nuclei from developing embryos (50% emergence) contain a predominant poly(A)+RNA component of approx. 5 . 10(5) Da. 125I-labelled preparations of nuclear DNA and of nuclear and membrane-associated poly(A)+RNA were used in reassociation and hybridization experiments with excess nuclear DNA. Poly(A)+RNA from the membranes of dormant cysts hybridized to nuclear DNA to the same extent as the nuclear poly(A)+RNA from developing embryos. The hybridization of labelled, nuclear poly(A)+RNA to nuclear DNA was strongly inhibited by unlabelled membrane RNA from either dormant cysts or developing embryos. It is concluded that the stored, membrane-associated poly(A)+RNA in dormant cysts is essentially of nuclear origin. The 5 . 10(5)-Da component is largely homologous with the corresponding component of nuclear poly(A)+RNA at later stages.

[Comparative study of cytoplasmic RNA-binding proteins of eukaryotic cells: content and polypeptide composition]. / Spavnitel'noe izuchenie tsitoplazmaticheskikh RNK-sviazyvaiushchikh belkov eukarioticheskikh kletok: soderzhanie i proipeptidnyi sostav.

The specific RNA-binding activity of cytoplasmic extracts of a number of eukaryotic cells and tissues was determined by sorption of radioactive RNA on nitrocellulose filters. This activity varies within a wide range: from 0,5 up to 80 microgram of 23S-RNA per mg of extract protein. The percentage of RNA-binding proteins as measured by their adsorption on RNA-Sepharose is 0,3--60% of total protein of cytoplasmic extracts and is well-correlated with their RNA-binding activity. The variability of the RNA-binding activity of the isolated preparations of RNA-binding proteins (from 100 to 300 microgram of 23S-RNA per mg of protein) is indicative of their qualitative differences. The analysis of the polypeptide composition of RNA-binding proteins revealed that the set of the major polypeptide chains is rather simple (with few exceptions) and includes both universal components with mol. weights of about 36,000, 49,000 and 95,000 characteristic of a number of cells and tissues and tissue- and species-specific polypeptides. Upon differentiation of rabbit reticulocytes into erythrocytes the percentage of RNA-binding proteins is decreased 4-fold and one of the three main polypeptides with the mol. weight of about 95,000 disappears. After fecundation of sea urchin ovicelles the polypeptide with the mol. weight of 37,000 disappears from the preparations of RNA-binding proteins and at the morula stage two new components with the mol. weights of about 35,000 and 26,000 appear instead of it. The germination of wheat embryos results in a two-fold increase of the percentage of RNA-binding proteins without any essential changes of the set of main polypeptide chains.

The light chain composition of embryonic myosin.

1. Myosins from an adult individual and embryos of Salmo trutta L. in different stages of development were isolated. Their light chain composition was investigated by polyacrylamide gel electrophoresis using two systems: tris-glycine buffer, pH 8.6, containing 8 M urea and 100 mM sodium phosphate buffer, pH 7.0, containing 0.1% SDS. 2. Both types of myosin are composed of three light chains with mol. wt 26200 D, 18300 D, 16800 D. 3. Almost no changes in electrophoretic patterns were discovered between the separate stages of development, except for the intensity of the light chain lc3, which increased gradually during miogenesis.

The molting hormones from the embryonated EGG of the tobacco hornworm, Manduca sexta (l.).

26-Hydroxyecdysone is the predominant molting hormone in 24- to 44-hour-old embryonated tobacco hornworm eggs, accounting for approximately 80% of the ecdysones present at this stage of development. This molting hormone was previously shown to be the major ecdysone present in 48- to 64-hour-old embryonated eggs of this insect. During both of these periods of embryonic development in the hornworm 20-hydroxyecdysone is a minor component, in contrast to its presence as the major ecdysone in the hornworm during certain stages of post-embryonic development.

Poly (A)-containing polyribosomal RNA in sea urchin embryos: changes in proportion during development.

Evidence is described which suggests that a large fraction of sea urchin embryo polyribosomal RNA is not polyadenylated, and that the proportion of poly(A)-containing RNA increases during embryological development. Cleavage, early blastula, and mesenchyme blastula stage embryos were treated with cordycepin, and the effects of the drug on polyribosome appearance were determined. Polyribosomes in cleaving embryos and early blastulae were unaffected by the drug. However, there was a significant reduction in the amount of polyribosomes present and in the amount of newly synthesized RNA entering polyribosomes in mesenchyme blastulae after cordycepin treatment. Polyribosomal RNA was isolated from cleavage, early blastula, and mesenchyme blastula stage embryos, and the RNA was characterized by sucrose gradient centrifugation. Cleaving embryo polyribosomal RNA has a prominent 9 S peak and a smaller 20 S peak, and the larger RNA is heterodisperse. There is no poly(A) associated with the 9 S and 20 S material, but there is poly(A) associated with the RNA sedimenting faster than 20 S. Early blastula and mesenchyme blastula polyribosomal RNA have 22 S and 26 S peaks, respectively. This later stage RNA is quite heterodisperse and poly(A) is associated with RNA in all size classes larger than 9 S. Finally, the proportion of polyadenylated light and heavy polyribosome RNA was determined for cleavage, early blastula, and mesenchyme blastula stage embryos. Approximately 20-25% of newly synthesized light polyribosome RNA is polyadenylated and this amount is relatively constant during early development. However, the proportion of newly synthesized RNA in heavy polyribosomes that is polyadenylated increases during development: 25% at cleavage, 33% at early blastula, and 48% at mesenchyme blastula stages.