Rabies mRNA translation in Xenopus laevis oocytes.

Two rabies virus-specific mRNA species were identified by analysis of their encoded proteins after translation of the partially purified species in Xenopus laevis oocytes. One of these coded for the virion surface glycoprotein (G protein), and the other coded for the major structural protein of the virion nucleocapsid (N protein). The G-mRNA sedimented in a sucrose density gradient at about 18S, and the N-mRNA had a sedimentation coefficient of approximately 16S. Their respective translation products were identified in a radioimmunoassay with specific monoclonal antibody probes that recognized only G or N proteins. Immunoprecipitates formed between the radiolabeled viral antigens synthesized in programmed oocytes and their respective monoclonal antibodies were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The glycoprotein antigen translated from G-mRNA in oocytes migrated in the gel ahead of the virion G protein with a migration rate that was similar to that of nonglycosylated intracellular glycoproteins from virus-infected cells. The results suggested that the branched-chain carbohydrate of G protein was not required for recognition by the particular monoclonal antibody used. The nucleocapsid antigen translated from N-mRNA in oocytes migrated to the same position in the gel as marker virion N protein. Both the electrophoretic mobility of virus-specific antigens in sodium dodecyl sulfate-polyacrylamide gel and the antibody concentration dependence for immunoprecipitations were criteria for identifying the individual viral proteins encoded by the two rabies mRNA's.     

The nucleotide sequence of the major beta-globin mRNA from Xenopus laevis.

The nucleotide sequence of a cloned fragment containing an almost complete copy of the mRNA encoding the major adult beta-globin polypeptide in Xenopus laevis, the South African Clawed Toad, is presented. A procedure for strand separation by hybridization to complementary mRNA was used to determine some of the sequence and this technique is described. The complete amino acid sequence of the polypeptide has been deduced and comparison with other vertebrate beta-globins reveals several highly conserved, and therefore potentially important, regions of the protein. The sequence of beta-globin mRNA has been determined in several mammals, and in the chicken. Thus we have searched for conserved regions in the non-coding portions of these mRNA sequences, which encode the same protein, but which have been evolving separately for several hundred million years.     

Export of mRNA from microinjected nuclei of Xenopus laevis oocytes

Export of mRNA from the nucleus to the cytoplasm was studied in mature Xenopus laevis oocytes. In vitro transcribed, capped 32P-labeled mRNA was microinjected into nuclei, and its appearance in the cytoplasm measured by counting radioactivity or by RNA extraction and gel electrophoresis. Both for a 5.0-kb transferrin receptor mRNA and a 2.0-kb 4F2 antigen heavy chain mRNA we found saturable transport with an apparent Km of 3.6 x 10(8) molecules per oocyte nucleus. Under non-saturating conditions the half-time for mRNA export from the nucleus was approximately 2 min at 20 degrees C. At higher concentrations of injected mRNA this half-time was prolonged, and the maximal transport rate was reached at approximately 1.6 x 10(8) molecules/min. mRNA transport showed properties of an energy-dependent mechanism, since it was inhibited at 4 degrees C or by ATP depletion. Co-injection of the cap dinucleotide m7GpppG blocked the export effectively, suggesting a role for the cap in this process. The export was also inhibited by the pre-injection of wheat germ agglutinin. The effect of the lectin was specific and abolished by co-injection of N-acetylglucosamine. Finally, we found significant competitive inhibition in mRNA export by the presence of tRNA. Our results suggest that mRNA transport is a facilitated process which may share common steps with tRNA transport. Preliminary gel retardation experiments show that injected mRNA associates with endogenous nuclear proteins and suggest an exchange of some of the bound components during the transport to the cytoplasm.     

Polyadenylated mRNA from the photosynthetic procaryote Rhodospirillum rubrum.

Total cellular RNA extracted from Rhodospirillum rubrum cultured in butyrate-containing medium under strict photosynthetic conditions to the stationary phase of growth has been fractionated on an oligodeoxythymidylic acid-cellulose column into polyadenylated [poly(A)+] RNA and poly(A)- RNA fractions. The poly(A)+ fraction was 9 to 10% of the total bulk RNA isolated. Analysis of the poly(A)+ RNA on a denaturing urea-polyacrylamide gel revealed four sharp bands of RNA distributed in heterodisperse fashion between 16S and 9S. Similar fractionation of the poly(A)- RNA resulted in the separation of 23, 16, and 5S rRNAs and 4S tRNA. Poly(A)+ fragments isolated after combined digestion with pancreatic A and T1 RNases and analysis by denaturing gel electrophoresis demonstrated two major components of 80 and 100 residues. Alkaline hydrolysis of the nuclease-resistant, purified residues showed AMP-rich nucleotides. Through the use of snake venom phosphodiesterase, poly(A) tracts were placed at the 3' end of poly(A)+ RNA. Stimulation of [3H]leucine incorporation into hot trichloroacetic acid-precipitable polypeptides in a cell-free system from wheat germ primed by the poly(A)+ RNA mixture was found to be 220-fold higher than that for poly(A)- RNAs (on a unit mass basis), a finding which demonstrated that poly(A)+ RNAs in R. rubrum are mRNAs. Gel electrophoretic analysis of the translation mixture revealed numerous 3H-labeled products including a major band (Mr, 52,000). The parent protein was precipitated by antibodies to ribulose bisphosphate carboxylase-oxygenase and comprised 6.5% of the total translation products.     

Heat production and respiration of the oocyte of Xenopus laevis L. in various stages of oogenesis]

The data are given on the respiration and heat production intensity in the X. laevis oocytes by which the value and patterns of change of psiu-function (function of bound dissipation) during oogenesis are calculated. All 3 values were shown to decrease rapidly from the stage of previtellogenesis till the stage of mature oocyte what agrees with the thermodynamic theory of development.     

cDNA cloning and developmental expression of fibroblast growth factor receptors from Xenopus laevis.

The heparin-binding growth factors constitute a family of homologous polypeptides including basic and acidic fibroblast growth factors (FGFs). These factors participate in a variety of processes, including wound healing, angiogenesis, neuronal survival, and inductive events in the early amphibian embryo. We have isolated three closely related species of cDNA clones for Xenopus FGF receptors. One of these, designated XFGFR-A1, encodes an open reading frame of 814 amino acids. A second class encodes an identical amino acid sequence with the exception of an 88-amino-acid deletion near the 5' end. This species probably arises through alternative splicing. A third class of cDNA corresponding to the shorter form of XFGFR-A1 was isolated and shown to be 95% homologous and is designated XFGFR-A2. Xenopus FGF receptors are similar to FGF receptors from other species in that they contain a transmembrane domain, a tyrosine kinase domain split by a 14-amino-acid insertion, and a unique conserved stretch of eight acidic residues in the extracellular domain. Overexpression of Xenopus FGF receptor protein by transfection of COS1 cells with the corresponding cDNA in a transient expression vector leads to the appearance of new FGF binding sites on transfected cells, consistent with these cDNAs encoding for FGF receptors. RNA gel blot analysis demonstrates that Xenopus FGF receptor mRNA is a maternal message and is expressed throughout early development. When blastula-stage ectoderm is cultured in control amphibian salt solutions, Xenopus FGF receptor mRNA declines to undetectable levels by late neurula stages. However, when cultured in the presence of FGF of XTC mesoderm-inducing factor, Xenopus FGF receptor RNA expression is maintained.     

FGF3 from Xenopus laevis.

Fibroblast growth factor 3 (FGF3) was first identified as the product of a cellular oncogene activated by mouse mammary tumour virus but its normal role appears to be in the developing embryo. To gain further insights into its function, we have isolated sequences encoding the FGF3 homologue in Xenopus laevis, XFGF3. COS-1 cells transfected with XFGF3 cDNA express a 31 kDa product, p31, generated by signal peptide cleavage and Asn-linked glycosylation at the single consensus site. This product is secreted and becomes associated with the cell surface and extracellular matrix. Proteolytic cleavage of p31 in the extracellular compartment results in an amino-terminally truncated product, p27, that is also glycosylated. Both p31 and p27 bind quantitatively to heparin-Sepharose and can be displaced from the cell surface and extracellular matrix by soluble heparin. Conditioned medium containing these two proteins is capable of inducing transient morphological transformation of NIH3T3 cells and of stimulating DNA synthesis in quiescent C57MG and BALB/MK cells which express different isoforms of FGF receptors 1 and 2. Since XFGF3 behaves very differently from its mouse counterpart, we constructed chimeras in which amino-terminal sequences from XFGF3 were fused with carboxy-terminal sequences from mouse FGF3. Increasing the contribution from mouse FGF3 led to a more restricted host range for the chimeric ligand.     

Translation "in vitro" of globin mRNA from Xenopus laevis

RNA isolated from Xenopus laevis reticulocytes and characterized as globin mRNA (Meza et al., 1978) was tested for its capacity to stimulate "in vitro" a wheat germ translation system, and the ability to synthesize a polypeptide. The latter was identified as globin by its electrophoretic mobility and immunoprecipitation with antiglobin antibody.     

Characterisation of bacterial clones containing DNA sequences derived from Xenopus laevis vitellogenin mRNA.

A 1700 nucleotide DNA sequence derived from Xenopus vitellogenin mRNA has been cloned in the bacterial plasmid pBR322. The identity of the cloned sequence was verified in two ways. Firstly, the plasmid DNA was shown to hybridise to an RNA of the correct size (6,700 nucleotides). This was shown by in situ hybridisation to electrophoretically separated RNA and also by the formation of "R-loops" with purified vitellogenin mRNA. Then, using a novel procedure in which plasmid DNA covalently bound to diazotised paper is used to select complementary mRNA sequences, the cloned sequence was shown to hybridise to an mRNA which directed the synthesis of vitellogenin when translated in a reticulocyte lysate cell-free system.     

Sequence analysis of 28S ribosomal DNA from the amphibian Xenopus laevis.

We have determined the complete nucleotide sequence of Xenopus laevis 28S rDNA (4110 bp). In order to locate evolutionarily conserved regions within rDNA, we compared the Xenopus 28S sequence to homologous rDNA sequences from yeast, Physarum, and E. coli. Numerous regions of sequence homology are dispersed throughout the entire length of rDNA from all four organisms. These conserved regions have a higher A + T base composition than the remainder of the rDNA. The Xenopus 28S rDNA has nine major areas of sequence inserted when compared to E. coli 23S rDNA. The total base composition of these inserts in Xenopus is 83% G + C, and is generally responsible for the high (66%) G + C content of Xenopus 28S rDNA as a whole. Although the length of the inserted sequences varies, the inserts are found in the same relative positions in yeast 26S, Physarum 26S, and Xenopus 28S rDNAs. In one insert there are 25 bases completely conserved between the various eukaryotes, suggesting that this area is important for eukaryotic ribosomes. The other inserts differ in sequence between species and may or may not play a functional role.