Differential Expression of Specific Proteins during In Vitro Tomato Organogenesis1

The cotyledons of tomato (Lycopersicon esculentum L., cv. Jia Fen-10) seedlings were induced to produce calli and regenerate plants via organogenesis. Utilizing this system, the composition and content of stage-specific proteins associated with organogenesis were analyzed. Moreover, a comparison of the protein composition and content between embryogenic and nonembryogenic calli was conducted. The SDS-PAGE results and laser densitometric scanning maps showed that there were different specific proteins expressed at different stages. Among them, six proteins (61, 54, 38, 37, 35, and 23 kD) were associated with the morphogenesis of organs, and two proteins (39 and 24 kD) were related to the morphogenesis of calli. Although no distinctive difference in protein components of embryogenic calli was noted, there were different trends of changes, both for the content of the proteins 39 and 24 kD, and for the content of the total proteins, at different developmental stages of embryogenic calli. The results obtained from the embryogenic and nonembryogenic calli indicated that these two materials were distinct in the protein components as well as in its content; for example, the protein 54 kD was detected in nonembryogenic but not in embryogenic calli. The total protein content in nonembryogenic calli was lower than that in the embryogenic calli.     

Cell-free translation of silkmoth chorion mRNAs: Identification of protein precursors and characterization of cloned DNAs by hybrid-selected translation

The secretory silkmoth chorion proteins are synthesized as precursors bearing signal peptides. Precursors are detected upon cell-free translation of chorion mRNAs in the wheat germ system; they are processed into products identical in size to authentic chorion proteins when translation is performed in the presence of microsomal membranes from dog pancreas. Precursors corresponding to specific protein size classes and subclasses are identified by three approaches: comparison of precursors and products encoded by stage-specific mRNAs, comparison of precursors and products encoded by mRNAs specifically hybridizing to individual chorion cDNA clones, and comparison of relative amino acid compositions of precursors and authentic chorion proteins. Translation of stage-specific mRNA preparations indicates that, in general, the developmental changes of in vivo chorion protein synthesis are based on changes in concentrations of the corresponding mRNAs. Characterization of the precursors makes it possible to identify, for any chorion DNA clone, the protein subclass, a member of which is encoded by the clone sequence.     

Stage-specific protein synthesis during early embryogenesis in Drosophila melanogaster

The changes in protein species synthesized during early Drosophila embryogenesis were characterized by two-dimensional electrophoresis. Of the 261 proteins scored, 68 (26%) show dramatic changes in rates of synthesis during the first 8 h of embryogenesis. These stage-specific proteins can be classified into three categories: early, detected at 1, 2 and 3 h but not later; late, not detected at 1 h, but appearing later; and discontinuous, detected before and after, but not at 3 and 4 h. RNA was extracted from three representative stages, translated in vitro, and the translation products separated on two-dimensional gels. There was a strong correlation between the patterns of synthesis in vivo and in vitro, suggesting that the early proteins are translated from maternal mRNA, and the late proteins from zygotic mRNA. A thorough comparison was made between the proteins synthesized in wild-type and dorsal embryos, in which virtually only dorsal hypoderm differentiates. The first observed difference was a reduced synthesis of actin I at 8 h, indicating that the absence of mesodermal and endodermal tissues is not detectable at the level of moderately abundant protein until the onset of differentiation.     

Adenovirus early gene products may control viral mRNA accumulation and translation in vivo

The mechanisms controlling early adenovirus gene expression in vivo have been studied using inhibitors of protein synthesis. When inhibitors were added shortly before or at the onset of infection, viral mRNA from all early regions was transcribed, spliced and accumulated over a 7 hr period. After longer pretreatment, accumulation of several early mRNAs were suppressed. Addition of inhibitors 1 hr after infection enhanced the accumulation of viral mRNA in the cytoplasm. Translation of early mRNA selected on adenovirus DNA in a cell-free system reflected the amount of viral mRNA present. A viral coded product may therefore control accumulation of viral mRNA.A different pattern emerged when inhibitors of protein synthesis were removed at 5 hr postinfection and cells were pulse-labeled in vivo. If inhibitors were introduced at or before infection, early viral proteins were synthesized only after a lag of 1–3 hr. However, if treatment was introduced 1 hr post-infection, reversion of the protein synthesis block was instantaneous. It appears that protein synthesis inhibitors reveal an in vivo translational block for viral mRNA. This block could be overcome by preinfection with a related virus. Furthermore, no block was observed in a virus-transformed human embryonic kidney cell line (293) which expresses early region 1 of the viral genome. Viral gene product(s) encoded in early region 1 may control translation of early adenovirus messenger RNA in vivo.     

Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm

Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non‐structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1‐4 does not block cellular protein synthesis in BHK cells. Trans‐complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co‐expression of nsP1‐4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T‐cell intracellular antigen and polypyrimidine tract‐binding protein is clearly detected in SINV‐infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut‐off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut‐off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR^?/? mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm.     

Developmental modulation of protein synthesis in Drosophila primary embryonic cell cultures

The patterns of proteins synthesized during embryonic development in Drosophila melanogaster have been examined by two-dimensional gel electrophoresis. Primary cell cultures prepared from donor embryos synchronized to ± 1 hr were labeled with [³⁵S]methionine at 5, 11.5, 14.5, and 26 hr after oviposition. Of approximately 400 to 500 proteins detected, the synthesis of about 50 is developmentally modulated. The greatest number of changes in the synthesis of stage-specific proteins occurs at 11.5 and 14.5 hr after oviposition, periods just prior to and during the times of the greatest overt morphological and biochemical changes. At 11.5 hr, 35 stage-specific proteins are synthesized, including 19 that are not present at the previous stage examined. At 14.5 hr, 34 stage-specific proteins can be detected, including 11 newly synthesized proteins. However, 12 proteins from the previous stage are no longer synthesized. At the completion of embryonic differentiation, at 26 hr, no new proteins are synthesized and the synthesis of many present in earlier stages has decreased or stopped. Comparison of patterns of embryonic proteins to those synthesized by two Drosophila continuous cell lines reveals that the majority of proteins are common to all. However, only about 40% of the embryonic stage-specific proteins are present in either cell line. In addition, there are several proteins unique to each cell line that are not observed in any of the embryonic stages.     

Acidic phosphoprotein complex of the 60S ribosomal subunit of maize seedling roots. Components and changes in response to flooding.

We determined that ribosomes of seedling roots of maize (Zea mays L.) contain the acidic phosphoproteins (P-proteins) known to form a flexible lateral stalk structure of the 60S subunit of eukaryotic ribosomes. The P-protein stalk, composed of P0, P1, and P2, interacts with elongation factors, mRNA, and tRNA during translation. Acidic proteins of 13 to 15.5 kD were released as a complex from ribosomes with 0.4 M NH4Cl/50% ethanol. Protein and cDNA sequence analysis confirmed that maize ribosomes contain one type of P1, two types of P2, and a fourth and novel P1/P2-type protein. This novel P-protein, designated P3, has the conserved C terminus of P1 and P2. P1, P2, and P3 are similar in deduced mass (11.4-12.2 kD) and isoelectric point (4.1-4.3). A 35.5- to 36-kD acidic protein was released at low levels from ribosomes with 1.0 M NH4Cl/50% ethanol and identified as P0. Labeling of roots with [32P]inorganic phosphate confirmed the in vivo phosphorylation of the P-proteins. Flooding caused dynamic changes in the P-protein complex, which affected the potential of ribosome-associated kinases and casein kinase II to phosphorylate the P-proteins. We discuss possible alterations of the ribosomal P-protein complex and consider that these changes may be involved in the selective translation of mRNA in flooded roots.     

A new extracellular matrix protein of the sea urchin embryo with properties of a substrate adhesion molecule

Summary A new embryonic extracellular matrix protein has been purified from eggs of the sea urchin Paracentrotus lividus. The molecule is a 210 kD dimer consisting of two 105 kD subunits that are held together by S-S bridges. In the unfertilized egg, the protein is found within granules uniformly distributed throughout the cytoplasm. After the egg is fertilized, the antigen is polarized to the apical surface of ectodermal and endodermal cells during all of the developmental stages examined, until the pluteus larva is formed. The protein promotes the adhesion of blastula cells to the substrate and is antigenically distinct from echinonectin, a well characterized substrate adhesion molecule. This report adds a new candidate to the list of known extracellular matrix molecules for the regulation of differentiation and morphogenesis in the sea urchin embryo. Offprint requests to: V. Matranga