Effect of diadenosine tetraphosphate microinjection on heat shock protein synthesis in Xenopus laevis oocytes.

Bisnucleosides polyphosphates are thought to be chemical messengers signalling to the cell the onset of various stresses. Diadenosine tri- and tetraphosphates (respectively, Ap3A and Ap4A) accumulate in prokaryotic and eukaryotic cells under heat shock conditions, suggesting they could trigger the synthesis of heat shock proteins (hsps). In this study, Ap4A, Ap3A and, as a control, Ap4 (adenosine tetraphosphate) were injected into Xenopus oocytes. Whereas none of these compounds is able to trigger the synthesis of hsps in the absence of hyperthermic treatment, nuclear microinjection of Ap4A after a mild heat shock specifically enhances the synthesis of the 70-kd hsp, which is involved in the regulation and possibly the termination of the heat shock response. The microinjection of Ap4A prior to the hyperthermic treatment results in a strong inhibition of hsps synthesis (with the exception of the 70-kd hsp) suggesting that Ap4A is involved in the regulation and/or termination of the heat shock response. Ap3A and Ap4 do not induce any detectable modification of hsps expression.     

Mitochondria DNA synthesis in oocytes of Xenopus laevis

The process of attachment was studied in primary mouse kidney epithelial cell cultures by means of reflexion contrast microscopy, a method developed for studying the cell membrane-substrate relationship. The first in a series of events is simple adherence to the substrate, called close contact. This phenomenon is associated with the greatest extension of lamellar cytoplasm and the fewest number of cell nuclei/unit area. The nuclei of such cells are in close contact with the bottom portion of the cell membrane. Approx. 24 h after planting, as the cultures become more crowded, cells develop a different kind of attachment to the substrate—focal contacts—that are correlated with a decrease in lamellar cytoplasm. Cells detached from the substrate after close contact formation readily reattach, while cells detached after formation of focal contacts do not reattach. After incubation for periods greater than 5 days, the dense cultures degenerate and cells lose their attachment to the glass surface.     

Minichromosome assembly accompanying repair-type DNA synthesis in Xenopus oocytes.

Minichromosomes were assembled by injection of circular DNA into the nucleus of Xenopus oocytes. We observed that, in the course of DNA supercoiling and chromatin assembly, a small percentage of the injected DNA molecules incorporated a radioactive precursor. This DNA synthesis was carried out by aphidicolin-sensitive DNA polymerase, and generated short repair-like patches covalently linked to the injected DNA. We found that the DNA thus repaired was rapidly supercoiled almost to completion within 15 to 30 min after injection, whereas 60 to 120 min were required to supercoil the intact, bulk DNA molecules. Such differential supercoiling kinetics was also observed when UV-damaged DNA was injected. Chromatin assembly, which was characterized by DNA fragment sizes protected from micrococcal nuclease digestion, was consistent with the rapid DNA supercoiling and proceeded more efficiently on the repaired DNA. These results indicate that there are at least two kinetically distinct ways of assembling minichromosomes in the oocyte nucleus, and that the repaired DNA molecules preferentially follow the faster pathway.     

Structural characterization of the proteins encoded by adenovirus early region 2A

Proteins encoded by adenovirus type 2 and type 5 early region 2A isolated from infected HeLa cells were compared to translation products of E2A-specific messenger RNA in a reticulocyte cell-free system and in Xenopus oocytes. The main cell-free translation product is a 72,000 Mr polypeptide which in HeLa cells as well as in Xenopus oocytes is converted into a 75,000 Mr phosphoprotein capable of binding to single-stranded DNA. Some minor proteins are proteolytic cleavage products of the major protein. In the cell-free system, three E2A polypeptides, 32,000, 37,000 and 44,000 Mr, are translated from minor polyadenylated mRNA species that can be separated from the major mRNA. Synthesis of all E2A polypeptides in vitro is inhibited by cap-analogs. The 44,000 Mr protein is also synthesized in Xenopus oocytes. Tryptic peptide maps of [35S]methionine-labeled E2A proteins were constructed using high pressure liquid chromatography and the position of the methionyl residues within each peptide was determined by amino acid sequencing procedures. This information and the DNA sequence of the adenovirus 5 E2A gene published by Kruijer et al. (1981) were used to align the peptides and to construct a map of the E2A proteins. Our data demonstrate that the major 75,000 Mr protein is coded for by a leftward reading frame of 529 amino acid residues located between 62 and 66 map units. The data also map six sites as targets for proteolytic enzymes. The minor E2A translation products have the same carboxy terminus as the major protein. The initiation codons of the 44,000, 37,000 and 32,000 Mr polypeptides probably correspond to amino acids 170, 243 or 244 and 290 of the major protein. Some functional properties of the major E2A protein are shared by the minor proteins and thus could be mapped. Major sites of phosphorylation, the region involved in binding to single-stranded DNA and the antigenic regions recognized by immune sera are located between amino acid residues 50 to 120, 170 to 470 and 170 to 240, respectively.     

An immunodominant domain in adenovirus type 2 early region 1A proteins.

Six independent rat hybridoma cell lines producing monoclonal antibodies to human subgroup C adenovirus early region 1A (E1A) proteins were isolated. Competition binding experiments revealed that each of the monoclonal antibodies was directed against the same epitope or overlapping cluster of epitopes on the E1A proteins. Viral E1A deletion mutants and deleted forms of E1A proteins expressed in Escherichia coli were used to localize the antibody recognition sites to sequences between amino acids 23 and 120, encoded within the first exon of the E1A gene. Similarly, polyclonal antisera raised against the trpE-E1A fusion protein, as well as against the native, biologically active E1A protein, were also directed primarily against this immunodominant region.     

Redundant control of adenovirus late gene expression by early region 4.

A series of human adenovirus type 5 derivatives carrying deletion mutations in early region 4 (E4) were constructed and characterized with respect to viral late protein synthesis, viral cytoplasmic late message accumulation, viral DNA accumulation, and plaquing ability. Viral late protein synthesis was essentially normal in cells infected by mutants expected to produce either the E4 open reading frame (ORF) 3 product or the E4 ORF 6 product. In cells infected by mutants lacking both ORF 3 and ORF 6, late protein synthesis was dramatically reduced. The basis for this reduction appears to be a concomitant reduction in cytoplasmic late message levels. Our results suggest that the products of ORFs 3 and 6 are redundant, since they are individually able to satisfy the requirement for E4 in late gene expression. Two of the mutants examined were defective for viral late protein synthesis but showed no measurable defect in viral DNA accumulation. The defect in late gene expression is not, therefore, a reflection of a primary defect in viral DNA synthesis. Finally, mutants expected to express ORF 3 or ORF 6 formed plaques with normal or only modestly reduced efficiency, whereas mutants expected to express neither ORF formed plaques with an efficiency less than 10(-6) that of wild-type virus. Thus, plaque-forming ability reflected late protein synthetic ability, suggesting that among these mutants late protein synthetic proficiency is the principle determinant of plaquing efficiency.     

Induction of transformation and DNA synthesis after microinjection of raf proteins.

Full-length and N-terminal deletion mutants of human c-raf-1 cDNA were cloned into Escherichia coli expression plasmids. Bacterially expressed c-raf proteins were purified by anion-exchange, gel filtration, and affinity chromatography. Microinjection of mutant c-raf proteins into G0-arrested NIH 3T3 cells induced DNA synthesis and morphological transformation, whereas microinjection of full-length c-raf had no effect. The amino terminus of the raf protein has an important negative regulatory influence; alteration of this region resulted in increased kinase activity and oncogenicity.     

Mitochondrial DNA synthesis in large and mature oocytes of Xenopus laevis

Deoxynucleosides are incorporated into mitochondrial DNA (mtDNA) of large oocytes; the rate of incorporation is about 2% of the mtDNA amount per 24 hr. When oocytes have been induced to mature in vitro with human chorionic gonadotropin (HCG), uptake and actual incorporation of thymidine decrease, although phosphorylation is enhanced. An examination of mtDNA replication shows that HCG treatment induces an increase in the relative synthesis of E-strands and an accumulation of D-loops. A similar effect is obtained by ethidium bromide treatment. Thus, gonadotropin appears to delay E-strand elongation and to synchronize mtDNA molecules at the begining of their replication cycle.     

Xenopus cyclin D2: Cloning and expression in oocytes and during early development

Summary— We have isolated and characterized a cDNA which contains the entire coding sequence of Xenopus laevis cyclin D2 protein. Cyclin D2 mRNA is identified as a member of the class of maternal RNAs. It is rare and stable during embryonic development at least until tadepole. In addition, a second cDNA coding for a truneated version of cyclin D2 was also isolated. Mieroinjection of cyclin D2 into oocytes undergoing meiotic maturation and parthenogenetic activation reveals that the protein is stable for several hours, independently of the ubiquitin-mediated degradation of cyclin B2 that takes place periodically during this process. Microinjected cyclin D2 localizes both in the cytoplasm and in the nucleus of oocyte. In somatic cells, it is well established that cyclin D2 is almost exclusively nuclear and very labile. The unusual behaviour of cyclin D2 upon injection into oocytes may provide indications about a possible role for this protein during meiosis and early development.     

Inhibition of DNA synthesis in living cells by microinjection of Gi2 antibodies.

Heterotrimeric guanine nucleotide binding proteins function in the coupling of a diverse span of cell surface receptors to a variety of intracellular signaling pathways, some of which stimulate cellular proliferation. With the recent discovery that mutated forms of G proteins are present in specific tumors, there has been an increased interest in the determination of the role of specific subtypes of G proteins in the regulation of cellular growth. We have attempted to determine which subtypes of G proteins are directly involved in serum-stimulated DNA synthesis through microinjection of inhibitory antibodies into living cells. Inhibitory rabbit polyclonal antibodies directed against specific Gi alpha subunits were introduced into living Balb/c 3T3 fibroblasts by microinjection, and the effect upon serum-stimulated DNA synthesis was examined. Results of these experiments indicate that Gi2 plays a direct role in serum-stimulated DNA synthesis in living cells and suggest that G proteins may function in a variety of mitogenic signaling pathways initiated by serum growth factors.     

Analysis of the intracellular localization and assembly of Ro ribonucleoprotein particles by microinjection into Xenopus laevis oocytes

Xenopus laevis oocytes have been used to determine the intracellular localization of components of Ro ribonucleoprotein particles (Ro RNPs) and to study the assembly of these RNA-protein complexes. Microinjection of the protein components of human Ro RNPs, i.e., La, Ro60, and Ro52, in X. laevis oocytes showed that all three proteins are able to enter the nucleus, albeit with different efficiencies. In contrast, the RNA components of human Ro RNPs (the Y RNAs) accumulate in the X. laevis cytoplasm upon injection. Localization studies performed at low temperatures indicated that both nuclear import of Ro RNP proteins and nuclear export of Y RNAs are mediated by active transport mechanisms. Immunoprecipitation experiments using monospecific anti-La and anti-Ro60 antibodies showed that the X. laevis La and Ro60 homologues were cross-reactive with the respective antibodies and that both X. laevis proteins were able to interact with human Y1 RNA. Further analyses indicated that: (a) association of X. laevis La and Ro60 with Y RNAs most likely takes place in the nucleus; (b) once formed, Ro RNPs are rapidly exported out of the nucleus; and (c) the association with La is lost during or shortly after nuclear export.     

Site-directed mutagenesis and expression of PC2 in microinjected Xenopus oocytes.

The biosynthesis and post-translational maturation of PC2, a neuroendocrine-specific Kex2-like endoprotease, following expression in Xenopus oocytes is described. The initial translation product was a 75-kDa membrane-associated protein which was released from the oocytes as a glycosylated 71-kDa protein. During extended chase periods, the extracellular 71-kDa protein was converted to a mature 68-kDa product. A deletion mutant lacking a putative COOH-terminal amphipathic helix was still membrane-associated, suggesting that this domain was not essential for attachment of PC2 to membranes. Two putative proregion cleavage site mutants were also constructed. Conversion of the 75-kDa peptide to the 71-kDa peptide involved cleavage at the sequence Lys-Arg-Arg-Arg (amino acids 78-81), since mutation of this sequence to Lys-Val-Arg-Leu resulted in the secretion of the 75-kDa peptide. Extracellular conversion of the 71-kDa peptide to the 68-kDa peptide involved cleavage at the sequence Arg-Lys-Lys-Arg (amino acids 106-109), since deletion of this tetrabasic sequence resulted in secretion of the 71-kDa peptide without further conversion to the 68-kDa form. Finally, a mutation which changed a catalytically important Asp to Asn did not affect processing of proPC2. These results may be relevant to our understanding of mechanisms in the intracellular sorting and maturation of proPC2 in neuroendocrine cells.     

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.