Biochemical research on oogenesis. Nucleotide sequence of initiator tRNA from oocytes and from somatic cells of Xenopus laevis.

The nucleotide sequence of initiator tRNA, tRNAfMet, from vitellogenic oocytes of Xenopus laevis was determined. The sequence was deduced from analysis of all T1 and pancreatic oligonucleotides and comparison with the sequence of initiator tRNA from other animal species. At least 80% of all initiator tRNA molecules from oocytes have the same nucleotide sequence. This means that most and probably all initiator tRNA genes which are active in oocytes are identical to one another. No structural difference was observed between liver and oocyte initiator tRNAs. Initiator tRNA from X. laevis has the same nucleotide sequence as initiator tRNA from several species of mammals. The genes coding for this RNA have therefore remained unchanged in the mammalian and amphibian lines for at least 300000000 years.     

Characterization of DNA-protein complexes from the mitochondria of Xenopus laevis oocytes

Mitochondrial DNA (mtDNA)-protein complexes (nucleoids) from Xenopus laevis oocytes were purified either on rate-zonal sucrose or isopyknic metrizamide gradients. From electron microscopic studies and staphylococcal nuclease digestion experiments mtDNA appears to be packaged into regular beaded structures. Protein electrophoretic analysis and M banding results show that mtDNA is associated with the membrane structures and also with few specific proteins including one acid-soluble polypeptide of 28 kD.     

Heterogeneous distribution and replication activity of mitochondria in Xenopus laevis oocytes

In early diplotene oocytes of Xenopus laevis mitochondria are not dispersed all over the cytoplasm but gathered in a well described mitochondrial mass [18]. When tracing these organelles during active vitellogenesis we observe that some of them are involved in the elaboration of a cortical layer at the vegetative hemisphere of the cell while others stay around the nucleus. The latter contribute to the transient formation of a mitochondrial crown throughout active mitochondriogenesis. Autoradiographic studies of thymidine incorporation into mtDNA suggest a differential participation of each organelle to the final population of a full-grown oocyte according to its position in the cytoplasm.     

Assembly of correctly spaced chromatin in a nuclear extract from Xenopus laevis oocytes.

Assembly of nucleosomes on relaxed, covalently closed DNA has been studied in a nuclear extract of Xenopus laevis oocytes. Nucleosomes containing the four histones H3, H4, H2A and H2B but lacking histone H1 are readily assembled on the DNA. The pattern of micrococcal nuclease digestion shows that the nucleosomes assembled in the absence of ATP and Mg (II) are closely packed, with a periodicity of 150 base pairs (bp). In contrast, in the presence of ATP and Mg (II) the spacing of the nucleosomes is 180 bp, similar to that observed for nucleosomes assembled on DNA microinjected into oocyte nuclei. The ATP and Mg (II) requirements for the assembly of correctly spaced nucleosomes are unrelated to the activity of the ATP and Mg (II) dependent DNA topoisomerase II in the extract; addition of specific inhibitors of eukaryotic DNA topoisomerase II has no effect on the spacing of the reconstituted nucleosomes. The ATP requirement in the assembly of correctly spaced nucleosomes can be substituted by adenosine 5'-O-3'-thiotriphosphate (gamma-S-ATP) but not by adenyl-5'-yl imidodiphosphate (AMP-P-(NH)-P).     

Export of proteins from oocytes of Xenopus laevis.

When human lymphoblastoid mRNA was microinjected into X. laevis oocytes, titers of interferon rapidly reached a maximum inside the oocyte while accumulation of interferon continued in the incubation medium for at least 45 hr. If interferon protein was injected into oocytes it was rapidly inactivated. Significantly, newly synthesized interferon but not injected interferon was found to be membrane-associated. Further experiments involving the co-injection of mRNAs coding for secretory proteins (guinea pig milk proteins and human interferon) and nonsecretory proteins (rabbit globin) revealed that only the secretory proteins were exported from the oocyte. Moreover, different proteins were exported at different rates. A distinct subclass of newly synthesized oocyte proteins of unknown function also accumulated in the incubation medium. Since the information encoded in the messenger RNAs of secretory proteins is sufficient to specify synthesis, compartmentation and secretion of these proteins, the oocyte may provide a complete system for the analysis of the secretory process.     

Aminoacyl transfer from phenylalanyl-tRNA microinjected into Xenopus laevis oocytes.

$\text{Xenopus laevis}$ oocytes were injected with [¹⁴C] phe-tRNA and the fate of the aminoacyl moiety was studied. The radioactive phenylalanine is gradually hydrolized off the tRNA once inside the cell. The rate of deacylation of the tRNA is not affected by inhibition of cellular protein synthesis by puromycin or cycloheximide. Part of the radioactive amino acid that leaves the tRNA (30 to 65%) is transferred directly into the oocyte nascent proteins as evidenced by the fact that its incorporation into proteins is not reduced by coinjection with a large excess of [¹²C] phenylalanine. Aminoacyl transfer from injected phe-tRNA into proteins is inhibited by puromycin and cycloheximide.     

Immunological localization of a major karyoskeletal protein in nucleoli of oocytes and somatic cells of Xenopus laevis

Oocyte nuclei of Xenopus laevis contain two major karyoskeletal proteins characterized by their resistance to extractions in high salt buffers and the detergent Triton X-100, i.e. a polypeptide of 68,000 mol wt which is located in the core complex-lamina structure and a polypeptide of 145,000 mol wt enriched in nucleolar fractions. Both proteins are also different by tryptic peptide maps and immunological determinants. Mouse antibodies were raised against insoluble karyoskeletal proteins from Xenopus oocytes and analyzed by immunoblotting procedures. Affinity purified antibodies were prepared using antigens bound to nitrocellulose paper. In immunofluorescence microscopy of Xenopus oocytes purified antibodies against the polypeptide of 145,000 mol wt showed strong staining of nucleoli, with higher concentration in the nucleolar cortex, and of smaller nucleoplasmic bodies. In various other cells including hepatocytes, Sertoli cells, spermatogonia, and cultured kidney epithelial cells antibody staining was localized in small subnucleolar granules. The results support the conclusion that this "insoluble" protein is a major nucleus-specific protein which is specifically associated with--and characteristic of--nucleoli and certain nucleolus-related nuclear bodies. It represents the first case of a positive localization of a karyoskeletal protein in the nuclear interior, i.e. away from the pore complex-lamina structure of the nuclear cortex.     

Synaptonemal complexes of Xenopus laevis.

Synaptonemal complexes (SCs) have been analyzed in spread Xenopus spermatocytes and oocytes. They showed all the usual features of animal SCs in addition to a high incidence of centromere mismatching. A centriole pair is visible throughout zygotene and pachytene. At zygotene the ends of SCs are markedly thickened and are clustered at the nuclear periphery.     

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.     

Amino acid uptake in Xenopus laevis oocytes.

The isolated oocytes from Xenopus laevis are able to take up radioactive amino acids from the exogenous medium. Most amino acids tested are taken up to reach concentrations higher than the extracellular medium. The initial uptake velocities vary with the external amino acid concentration in a Michaelis-Menten fashion. Aspartic acid requires concentrations an order of magnitude higher than the five other amino acids tested to reach half the maximal uptake velocity. The uptake mechanism seems to be specific for groups of analogous amino acids, as can be determined by competition studies. The amino acid groups for which there is some evidence of uptake specificity would be aromatic, aliphatic, acidic and basic. Amino acid pools of oocytes show that these cells can concentrate amino acids from Xenopus blood, as well as from artificial media.     

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.