In vitro incorporation of amino acids into proteins stimulated by RNA from unfertilized sea urchin eggs

Maggio, Rachele; Vittorelli, Maria Letizia; Rinaldi, Anna Maria; Monroy, Alberto

doi:10.1016/0006-291x(64)90481-4

Cited by 64 publications

(11 citation statements)

References 14 publications

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“…This was originally based on the observation that protein synthesis following fertilization is not inhibited by actinomycin-D (Gross et al, 1964). It has been demonstrated that the stored mRNA in the sea urchin embryo is capable of being translated efficiently in vitro (Maggio et al, 1964). Likewise, the ungerminated spores of O. sensibilis have been shown to store large quantities of mRNA, and this stored mRNA is capable of being translated in vitro, for the synthesis of germination-specific proteins (Raghavan, 1987(Raghavan, , 1991.…”

Section: Discussionmentioning

confidence: 99%

Spermiogenesis inMarsilea vestita: A temporal correlation between centrin expression and blepharoplast differentiation

Hart

Wolniak

1998

Cell Motil. Cytoskeleton

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Section: Discussionmentioning

confidence: 99%

Spermiogenesis inMarsilea vestita: A temporal correlation between centrin expression and blepharoplast differentiation

Hart

Wolniak

1998

Cell Motil. Cytoskeleton

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“…Since actinomycin D does not inhibit the incorporation of leucine-3H into crayfish oocyte proteins, it seems likely that this synthesis is not dependent upon the immediate synthesis of RNA ; a condition which suggests that a fairly long-lived messenger RNA is present in the crayfish oocyte . The existence of template-active RNA in unfertilized amphibian and echinoderm oocytes has been established (11,30,40) . Furthermore, Electron microscope radioautographs of oocytes incubated in vivo with leucine-3H .…”

Section: Discussionmentioning

confidence: 99%

“…By 3 days, label occurs over forming yolk bodies (FY in Figs . 27 FIGURES [30][31][32] Portion of oocytes cultured with leucine-3H for 19 days under in vivo conditions . By this time most of the label is localized over yolk spheres (YB), but a few silver grains still occur over branching elements of endoplasmic reticulum (ER in Fig .…”

Section: Discussionmentioning

confidence: 99%

INTRACELLULAR SYNTHESIS, TRANSPORT, AND PACKAGING OF PROTEINACEOUS YOLK IN OOCYTES OF ORCONECTES IMMUNIS

Ganion

Kessel

1972

The Journal of Cell Biology

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The incorporation of leucine-3 H into either ovarian or oocyte proteins occurs throughout vitellogenesis, but is at a maximum during early phases of this process . The labeling of ovarian and oocyte proteins is inhibited with cycloheximide . Oocytes are permeable to actinomycin D, and this drug does not affect the incorporation of amino acids into oocyte proteins but does block oocyte RNA synthesis . By means of both light microscope and high resolution radioautography, it has been demonstrated that the initial incorporation of leucine-3 H under both in vitro and in vivo conditions occurs in elements of the roughsurfaced endoplasmic reticulum in the oocyte . Under pulse-chase conditions, the label subsequently becomes associated with intracisternal (precursor yolk) granules now aggregated within the cisternae of the connected smooth-surfaced endoplasmic reticulum . By 7 days, mature yolk globules are extensively labeled . The results of experiments designed to assess the possible contribution of maternal blood proteins to yolk deposition indicate that such a contribution is minimal . It is concluded that the crayfish oocyte is programmed for and capable of synthesizing the massive store of proteinaceous yolk present in the egg at the end of oogenesis . 420

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“…Several attempts have been made to demonstrate directly the presence of maternal mRNA in eggs of echinoid (2,(6)(7)(8)(9)(10) and amphibian (11) origin and in ungerminated plant embryos (12). In no case, however, has an RNA fraction been isolated and used to direct in vitro the synthesis of an identifiable product.…”

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confidence: 99%

Cell-Free Translation of Maternal Messenger RNA from Sea Urchin Eggs

Gross

Jacobs-Lorena

Baglioni

et al. 1973

Proc. Natl. Acad. Sci. U.S.A.

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The template activity of RNA extracted from unfertilized sea urchin eggs was demonstrated in a cell-free translation system, and, for the first time, specific proteins were identified among the products. All messenger RNA activity is recovered, under the conditions used, from the postribosomal supernatant. Histones, among many other proteins, were identified specifically as products of this "maternal" messenger RNA.Messenger RNA synthesized during oogenesis ("maternal mRNA") is used in sea urchin and other embryos to direct protein synthesis during development (1, 2). New mRNA is transcribed and used soon after fertilization, but transcription blockade by actinomycin D permits many cleavages and a long period of at least superficially normal development, to the hatched blastula stage in sea urchin. Transcription-blocked embryos continue to synthesize proteins actively during this time (3). Indirect evidence favors the storage of maternal mRNA in the cytoplasm (4) and the increasing availability of this RNA for translation as development proceeds (5). Availability has not been defined in chemical terms.Several attempts have been made to demonstrate directly the presence of maternal mRNA in eggs of echinoid (2, 6-10) and amphibian (11) origin and in ungerminated plant embryos (12). In no case, however, has an RNA fraction been isolated and used to direct in vitro the synthesis of an identifiable product.Spirin proposed early that mRNA may be associated with specific proteins in ribonucleoprotein particles he named "informosomes" (13 MATERIALS AND METHODSFractionation of Sea Urchin Homogenate. Eggs were collected from sea urchins Lytechinus pictus as described (21). 16 ml of pooled eggs was washed five times with Milliporefiltered sea water at 16-18°, twice with ice-cold Ca-and Mgfree sea water (37), and once with ice-cold 0.2 M KCI-5 mMI MgCl2-20 mM Tris buffer, pH 7.6 (TKM). All subsequent operations were performed at 0-4°. The medium chosen maintains best the integrity of embryonic polyribosomes (22). The eggs were resuspended in four volumes of TKM buffer containing 1 mg/ml of bentonite and homogenized in a Dounce glass homogenizer with a loose pestle. The homogenate was fractionated by successive centrifugations. Pellets were obtained by a choice of centrifugation conditions that separated eggs homogenized in an isotonic medium into unbroken cells and nuclei (1.5 min, 200 X g), pigment granules (10 min, 1500 X g), yolk granules (10 min, 5000 X g), and mitochondria plus microsomes (15 min, 12,000 X g). 5.0 ml of the 12,000 X g l)ostmitochondrial supernatant was layered over 32.5 ml of a 20-40% sucrose gradient in TKM buffer and centrifuged 19 hr at 26,000 rpm in an SW 27 rotor. The gradients were monitored for A260 in a Gilford recording spectrophotometer, and fractions were pooled as shown in Fig. l. RNA Extraction and Fractionation. Pellets obtained by differential centrifugation and that obtained from the gradient of Fig. 1 were resuspended in Na dodecyl sulfate-buffer at room temperature (230). Gra...

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In vitro incorporation of amino acids into proteins stimulated by RNA from unfertilized sea urchin eggs

Cited by 64 publications

References 14 publications

Spermiogenesis inMarsilea vestita: A temporal correlation between centrin expression and blepharoplast differentiation

Spermiogenesis inMarsilea vestita: A temporal correlation between centrin expression and blepharoplast differentiation

INTRACELLULAR SYNTHESIS, TRANSPORT, AND PACKAGING OF PROTEINACEOUS YOLK IN OOCYTES OF ORCONECTES IMMUNIS

Cell-Free Translation of Maternal Messenger RNA from Sea Urchin Eggs

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