Very long-lived messenger RNA in ovaries of Xenopus laevis

It is possible to label with radioactivity newly synthesized ovarian RNA after intraperitoneal injection of [³H]guanosine and [³H]uridine into immature Xenopus laevis, if ovaries in which only previtellogenic stage 1 oocytes are present. Following the amount of radioactivity in the ovarian pool of acid-soluble precursors indicates a complete clearance of acid-soluble radioactivity within 15–20 days after injection. Incorporation of radioactivity into total RNA (which is almost exclusively 4 and 5S RNAs at this stage) and poly(A)⁺ RNA ceases between 15 and 20 days after injection, but the total amount of radioactivity in these RNA fractions does not decline appreciably over the next 18 months. During this time, the ovary grows and develops since stage 6 oocytes eventually appear and there is a 10- to 20-fold increase in total RNA content, which changes in composition from almost exclusively (95%) 4 and 5S RNAs to mainly (75%) 18 and 28S RNAs. Thus, despite continued growth and development, radioactive RNA molecules synthesized during previtellogenesis survive for lengths of time commensurate with the length of oogenesis (1–2 years). Although very limited (<7%) reincorporation of radioactivity into RNA is detected, it cannot alone account for the stability of the label in poly(A)⁺ RNA. These results are interpreted as indicative of synthesis during previtellogenesis of tRNA, 5SrRNA, and messenger RNA molecules which are very long-lived.     

Persistence of nonribosome bound 5 S RNA in full-grown oocytes of Xenopus laevis

The 4 and 5 S RNA containing 42 S ribonucleoprotein (RNP) particles characteristic of previtellogenic and white oocytes cannot be detected in full-grown oocytes. When full-grown oocyte RNPs are separated on sucrose gradients 4 and 5 S RNA cannot be detected in the 42 S region. However, not all of the 5 S RNA stored during early oogenesis is incorporated into ribosomes at later stages. A substantial pool (20% of the total) of 5 S RNA remains in a non-ribosome-bound fraction sedimenting at about 7 S in full-grown oocytes.     

Patterns of protein synthesis and metabolism during sea urchin embryogenesis

We have analyzed the patterns of protein synthesis in developing embryos of the sea urchin Strongylocentrotus purpuratus by two-dimensional gel electrophoresis. There was an increase in the number of proteins detectably synthesized during development, as well as significant changes in relative rates of synthesis involving approximately 20% of the nearly 900 newly synthesized polypeptides. The majority of these changes were increases rather than decreases in synthesis; about half were of at least 10-fold, while a few were of more than 100-fold. Very few changes were detected upon fertilization and during the first several hours of development, while about 60% of the changes detected occurred between the hatching and the beginning of invagination. An analysis of proteins detected by silver staining indicated that most remained nearly constant in mass during embryonic development, but several increased or declined substantially. Many proteins present in eggs were not detectably synthesized in either eggs or embryos.     

Classification of DNA polymerase activities from ovaries of the frog,Xenopus laevis

Four distinct DNA polymerase activities were isolated from ovaries of the frog Xenopus laevis. Specific assays for each activity were established. The isolated activities were characterized by molecular weight, template-primer preferences, and sensitivity to specific inhibitors as Xenopus laevis ovarian DNA polymerases-α₁, -α₂, -β, and -γ. All previously described Xenopus laevis DNA polymerases were classified using these properties.     

Mitochondrial DNA-binding proteins that bind preferentially to supercoiled molecules containing the D-loop region of Xenopus laevis mtDNA

From the bulk of the Xenopus laevis mitochondrial proteins insoluble in 1% Triton X-100 + 1M NaCl, we have isolated, by DNA-cellulose chromatography, a protein fraction enriched in DNA-binding proteins. This fraction contains proteins showing a specific affinity for supercoiled DNA molecules containing the mitochondrial DNA displacement-loop region, as measured by filter binding and competition assays.     

Differential mRNA accumulation and translation during Spisula development

The patterns of proteins synthesized in developing Spisula embryos and larvae were compared with in vitro translation products by one-dimensional gel electrophoresis. Major changes in the in vivo pattern occur at fertilization; these are regulated at the translational level (Rosenthal, Hunt, and Ruderman, 1980, Cell 20, 487-494). The pattern is further altered by midcleavage, and subsequent development is accompanied by frequent changes in the kinds of proteins made. By midcleavage many of the in vivo changes are paralleled by alterations in mRNA levels. Three cDNA clones containing developmentally regulated, nonmitochondrial sequences were isolated from a library constructed from veliger larval RNA. Clone 3v4 encodes alpha-tubulin. Clone 12v4 encodes a 35,000-D protein of unknown function. The protein product of clone 10v8 has not been identified. The concentration of alpha-tubulin RNA is relatively low through midcleavage, increases by the swimming gastrula stage, and is maintained at a moderately high level throughout larval development. 10v8 and 12v4 RNAs first appear in trochophore larvae; their concentrations peak 10-12 hr later, and then decline. The proportions of alpha-tubulin and 10v8 RNA that are translated vary with developmental stage. During early cleavage very little alpha-tubulin RNA is on polysomes; in swimming gastrulae 64% of this mRNA is polysomal. Seventy percent of 10v8 RNA is translated in the trochophore larva, while only approximately 40% is polysomal in the 21-hr veliger. These results show that translational regulation may be superimposed on changes in cytoplasmic mRNA concentrations to determine the level of gene expression during embryogenesis.     

Regulation of light-harvesting chlorophyll-binding protein (LHCP) mRNA accumulation during the cell cycle in Chlamydomonas reinhardi

Light-harvesting chlorophyll a/b protein (LHCP) synthesis is highly regulated during the cell cycle in light-dark synchronized C. reinhardi cells. LHCPs are a family of cytoplasmically synthesized proteins which are imported into the chloroplast. LHCPs are derived from at least two precursor proteins (32 kd and 30 kd) that are synthesized in vitro and immunoprecipitated by antiserum against chlorophyll-protein complex II proteins. A DNA copy of the mRNA encoding a 32 kd LHCP precursor was cloned from cDNA synthesized from poly(A) RNA obtained from mid-light-phase synchronous cells. Using cloned cDNA (pHS16) as a hybridization probe, we found that a single 1.2 kb RNA complementary to pHS16 accumulates in a wave-like manner during the mid-light phase of the 12 hr light-12 hr dark cycle and correlates with the pattern of chlorophyll synthesis. Light, during the light phase in the light-dark cycle, is required for accumulation of this RNA.     

Intermediate filament protein synthesis in preimplantation murine embryos

The synthesis of two extraembryonic endodermal cytoskeletal proteins (Endo B, Mr = 50,000; Endo A, Mr = 55,000) was detected by immunoprecipitation at the 4- to 8-cell stage of preimplantation mouse development. The first detectable synthesis of both proteins occurs at about the same time as the earliest allocation of cells to the trophectodermal lineage. Both Endo A and B were identified in the two-dimensional gel pattern of blastocyst cytoskeletal proteins prepared by nonionic detergent and high-salt extraction. Endo A and B were identified as the y and x blastocyst cytoskeletal proteins, respectively, previously described by other investigators. Antibodies to Endo B are shown to react with intermediate filaments at the electron microscopic level, confirming that Endo B is an authentic intermediate filament protein. Previously, the TROMA 1 monoclonal antibody prepared by other investigators was shown to react specifically with Endo A and to decorate trophoblast cytoskeletons but did not react with the inner cell mass of blastocysts. Endo B antibodies are now also shown to decorate trophoblast cytoskeletons.     

Haploid accumulation and translational control of phosphoglycerate kinase-2 messenger RNA during mouse spermatogenesis

The intracellular location of the mRNA for the testis-specific isozyme of phosphoglycerate kinase-2 (PGK-2) has been determined for two spermatogenic cell types. The mRNA activity for PGK-2 from the polysomal and nonpolysomal fractions of pachytene primary spermatocytes or round spermatids has been assayed by cell-free translation with the polypeptide products monitored by immunoprecipitation, followed by one-dimensional or two-dimensional electrophoresis and fluorography. The results reveal that the majority of PGK-2 mRNA activity of round spermatids was present in the polysomal fraction while the relatively less abundant PGK-2 mRNA of pachytene primary spermatocytes was present in the nonpolysomal fraction. No PGK-2 mRNA activity was observed in the cytoplasmic RNA from primitive type A spermatogonia or prepubertal Sertoli cells. These data indicate that mature PGK-2 mRNA first appears in the cytoplasm of spermatogenic cells during the prophase of meiosis and increases in amount after meiosis. Although mature PGK-2 mRNA is present in meiotic cells it is not actively translated until after meiosis has been completed. Thus, mRNA accumulation and translational mechanisms are involved in the control of phosphoglycerate kinase-2 synthesis during spermatogenesis.     

Patterns of protein synthesis in livers of Xenopus laevis during metamorphosis: Effects of estrogen in normal and thyrostatic animals

Two-dimensional gel electrophoresis has been used to analyse protein synthesis in the livers of Xenopus laevis larvae during metamorphosis. The patterns found at different developmental stages have been characterised and compared to those found in developmentally static tadpoles and estrogen-treated tadpoles. The results suggest that the majority of proteins synthesized by the larval liver during metamorphosis can be divided equally into three main categories: those which are synthesized continuously, those whose synthesis is lost, and those whose synthesis is gained during development. The synthesis of proteins tends to be lost earlier in metamorphosis than it is gained. The pattern of liver protein synthesis in thyrostatic animals is not characteristic of any single stage of normal development, and displays features characteristic of many different stages. About half the changes in protein synthesis which occur during normal metamorphosis are dependent upon it. All the stages examined are responsive to estrogen, and each has a characteristic response. Half of the estrogen-induced changes in protein synthesis are independent of metamorphosis, while the other half require metamorphosis.     

The onset of activation responsiveness during maturation coincides with the formation of the cortical endoplasmic reticulum in oocytes of Xenopus laevis

Immature, Stage VI oocytes of Xenopus laevis fail to activate (i.e., to propagate a cortical reaction and elevate a fertilization envelope) when pricked or exposed to A23187. We determined the times during maturation when immature oocytes treated with progesterone in vitro developed the capacity to respond to pricking and to ionophore. Responsiveness to ionophore first appears at about 3.5-4.5 hr after progesterone treatment; all oocytes are activated by 8-9 hr after progesterone. The capacity to respond to pricking appears about 1.0-1.5 hr after first signs of ionophore responsiveness. We examined the cortical endoplasmic reticulum (CER) by TEM to determine whether the morphology of this component could be correlated with the development of responsiveness during maturation. Fully mature oocytes exhibit an extensive CER that (1) forms a "shell" around most cortical granules, (2) appears to interconnect cortical granules, and (3) forms junctions with the plasma membrane. The CER-plasma membrane junctions are especially obvious in preparations of isolated cortex. The elaborate CER is not present in immature oocytes. It first appears during maturation of progesterone-treated oocytes at 4.5-5.0 hr, coincident with the time when maturing oocytes develop their responsiveness to ionophore and to pricking. This temporal correlation is consistent with the hypothesis that the CER is one of the components required for regulation of intracellular free calcium in oocytes.     

Mapping of mitochondrial 4 S RNA genes in Xenopus laevis by electron microscopy

The distribution of sites hybridizing with mitochondrial 4 S RNA molecules on mitochondrial DNA of Xenopus laevis has been mapped in relation to the ribosomal RNA genes and EcoRI restriction endonuclease sites. RNA molecules linked to ferritin were employed for this purpose. We have obtained evidence for 15 4 S RNA sites on the H-strand and six sites on the L-strand of X. laevis mtDNA^∥. An indication of the possible existence of one additional site on the H-strand and four additional sites on the L-strand has been obtained. One 4 S RNA site is located in the gap between the two rRNA genes, and one site flanks each outside end of the rRNA genes. The other 4 S RNA sites are distributed almost evenly throughout both strands of the mtDNA. A comparison with the map of 4 S RNA sites on the mtDNA of HeLa cells (Angerer et al., 1976) suggests considerable evolutionary conservation of site organization.     

Interaction of wheat protein synthesis elongation factor 1 with GTP analogs

Wheat protein synthesis elongation factor 1 was tested for binding to GTP analogs, including structures resembling “caps” that are present at the 5′-termini of most eukaryotic mRNAs. The interaction was assayed by determining the capacity of the analogs to inhibit the binding of [³H]GTP to elongation factor 1. Significant interaction of elongation factor 1 with G(5′)ppp(5′)G, G(5′)pppp(5′)G, and G(5′)ppp(5′)A was observed. Methylation of a ribose 2′-hydroxyl had very little effect, but methylation of the 7 position of guanosine greatly diminished the affinity of elongation factor 1 for these compounds. m⁷G(5′)ppp(5′)Cm, m⁷G(5′)ppp(5′)Um, and m⁷G(5′)ppp(5′)Am gave no detectable binding with EF1.     

Cell-specific regulation of protein synthesis in the sea urchin gastrula: A two-dimensional electrophoretic study

Protein synthesis in the two cell types of echinoid early gastrulae was analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography. Epithelial cells and primary mesenchyme cells were isolated from early gastrulae as described by M. A. Harkey and A. H. Whiteley, 1980 (Wilhelm. Roux's. Arch.189, 111–112). Newly synthesized proteins were labeled with [³H]valine, extracted in SDS buffer, and analyzed electrophoretically. Of the 454 labeled proteins analyzed, 58 incorporated [³H]valine at markedly different relative rates in the two cell types, and 69 were labeled exclusively in one or the other cell type. The most rapidly synthesized proteins in gastrula cells constituted a class which exhibited a much higher degree of cell specificity than the total protein population. Several of these rapidly synthesized proteins were analyzed individually. Among those that were synthesized preferentially in primary mesenchyme cells, two low-molecular-weight, acidic proteins, designated PM28 and PM32, accounted for 9–14% of the total protein synthesis in primary mesenchyme cells but were barely detectable in epithelial cells. Those proteins that were synthesized preferentially in the epithelial cells included several low-molecular-weight species, probably histones, and the cytoskeletal proteins, actin and tubulin. These data indicate that the primary mesenchyme and epithelium of the early gastrula differ profoundly with respect to the synthesis of specific proteins.     

Mouse interferon messenger RNA: characterization of the Xenopus laevis oocyte translation product

Mouse interferon messenger RNA was isolated from Newcastle disease virus-induced mouse Lpa cells and then translated in Xenopus laevis oocytes. The resulting oocyte homogenate containing translated interferon activity was unstable to treatment with 5 m urea and to repeated freeze/thaw cycles, and it was 1% cross-reactive on human cells, as was native mouse interferon. Both native mouse interferon and the mouse interferon produced by the translation of mouse interferon mRNA behaved almost similarly on CPG, poly(U)-Sepharose, and anti-mouse interferon antibody columns. When the oocyte-translated product was partially purified and analyzed on sodium dodecyl sulfate-polyacrylamide gels, it migrated as a major single band of activity at 21–22,000 daltons with a trailing edge at 22–30,000 daltons. Only minor activity was detected in the region of 35–40,000 daltons where the vast majority of the native mouse interferon migrated. Thus, the oocyte-translated mouse interferon product comigrated largely with the minor species of native mouse interferon with a little activity which corresponds with the larger molecular weight species of native mouse interferon.     

Preferential expression of actin genes during oogenesis of Drosophila

The expression of actin genes was examined during oogenesis of Drosophila. Accumulation of actin proteins was quantitated by a two-stage electrophoresis procedure. Egg chambers accumulate actins preferentially, resulting in a twofold enrichment over other nonyolk proteins. RNA gel blot hybridization experiments demonstrated a concomitant twofold selective increase of actin mRNA levels over that of other mRNAs, suggesting regulation of actin genes at the pretranslational level. Despite an abrupt arrest of actin protein accumulation near the end of oogenesis, the bulk of the actin mRNAs remains associated with polysomes of constant size. It appears that this shut-off of actin protein accumulation is due to an overall decrease in translational efficiency, rather than actin mRNA degradation or its dissociation from polysomes.     

Stimulation of protein phosphorylation during fertilization-induced maturation of Urechis caupo oocytes

Protein phosphorylation was studied during fertilization of Urechis caupo oocytes both in vivo, by measuring [³²P]phosphate incorporation into ³²P preloaded oocytes and in vitro, by measuring endogenous protein kinase and phosphatase activities in homogenates. During fertilization (and maturation) the rate of protein phosphorylation is dramatically increased. No change in the [³²P]phosphate uptake, or the nucleotide levels was observed at fertilization, so the increase cannot be attributed to changes in substrate availability. In vitro enzyme assays showed changes in protein kinase activity which approximately mirrored the changes in the in vivo phosphorylation pattern. As there were no changes in protein phosphatase activity, these results suggest the phosphorylation change results from an increase in protein kinase activity. The pattern of change, investigated by SDS-polyacrylamide gel electrophoresis, shows that proteins that were phosphorylated in the unfertilized egg become phosphorylated to a greater degree after fertilization. One protein (48 kd) undergoes an increase followed by a decrease of its phosphorylation level.     

Single-stranded bacteriophage T5 stO DNA as template for in vitro fMet-tRNA binding to ribosomes and protein synthesis.

Good evidence is provided that fMet-tRNA binding and aminoacid incorporation, when single-stranded DNA is used instead of mRNA in an E. coli cell-free system, are strictly dependent on the magnesium concentration. Ten sites homologous to the initiation sites of translation can be detected on denatured T5 stO DNA when using ribosomes and initiation factors from uninfected E. coli F. In S-30 extracts, at high magnesium concentrations and in the presence of neomycin, initiation of the translation of denatured T5 stO DNA begins anywhere on the molecule, and yet high molecular weight polypeptides are synthesized. The template potentiality of the denatured T5 stO DNA decreased when using ribosomes plus initiation factors and crude extracts from T5 stO-infected bacteria. By in vitro formation of initiation complexes sites analogous to initiation sites of translation were localized on T5 stO DNA molecules using single-stranded fragments separated by sedimentation in alkaline sucrose gradient.