Alan Colman

Identification of the sequence responsible for the nuclear accumulation of the influenza virus nucleoprotein in Xenopus oocytes

Influenza virus nucleoprotein (NP), synthesized in Xenopus oocytes after injection of cloned NP cDNA, enters and accumulates in the nucleus. We have used in vitro mutagenesis of this cDNA to study the cellular distribution of mutated NP polypeptides. Mutants lacking amino acids 327-345 of wild-type NP enter the nucleus but do not accumulate there to the same extent as the wild-type protein, suggesting that this region has a role in nuclear accumulation. This possibility is further strengthened by similar studies involving the production of fusion proteins in which various amino-terminal sequences of the NP gene are fused to the complete chimpanzee alpha 1-globin sequence: when globin cDNA was injected into and expressed in oocytes the protein remains exclusively in the cytosol; however, when the globin cDNA is fused to a portion of NP cDNA that includes the region encoding amino acids 327-345, the resulting fusion protein enters and accumulates in the nucleus. Fusion proteins lacking this region of the NP enter but do not accumulate in the nucleus.

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.

Segregation of mutant ovalbumins and ovalbumin-globin fusion proteins in Xenopus oocytes: Identification of an ovalbumin signal sequence

The intramolecular signals for chicken ovalbumin secretion were examined by producing mutant proteins in Xenopus oocytes. An ovalbumin complementary DNA clone was manipulated in vitro, and constructs containing altered protein-coding sequences and either the simian virus 40 (SV40) early promoter or Herpes simplex thymidine kinase promoter, were microinjected into Xenopus laevis oocytes. The removal of the eight extreme N-terminal amino acids of ovalbumin had no effect on the segregation of ovalbumin with oocyte membranes nor on its secretion. A protein lacking amino acids 2 to 21 was sequestered in the endoplasmic reticulum but remained strongly associated with the oocyte membranes rather than being secreted. Removal of amino acids 231 to 279, a region previously reported to have membrane-insertion function, resulted in a protein that also entered the endoplasmic reticulum but was not secreted. Hybrid proteins containing at their N terminus amino acids 9 to 41 or 22 to 41 of ovalbumin fused to the complete chimpanzee alpha-globin polypeptide were also sequestered by oocyte membranes. We conclude that the ovalbumin signal sequence is internally located within amino acids 22 to 41, and we speculate that amino acids 9 to 21 could be important for the completion of ovalbumin translocation through membranes.

Efficient expression of cloned complementary DNAs for secretory proteins after injection into Xenopus oocytes

Cloned complementary DNAs encoding chicken ovalbumin, chicken prelysozyme and calf preprochymosin, prochymosin and chymosin were inserted downstream from various viral promoters in modified recombinant shuttle vectors. Microinjection of the ovalbumin, prelysozyme and preprochymosin constructs into the nuclei of Xenopus laevis oocytes resulted in the synthesis, segregation in membranes and secretion into the extracellular medium of ovalbumin, lysozyme and prochymosin, respectively. Judging from molecular weight estimations, lysozyme and prochymosin were correctly proteolytically processed while ovalbumin, which lacks a cleavable signal sequence, was glycosylated. Injection of the DNA construct encoding prochymosin without its signal sequence resulted in synthesis of prochymosin protein that was localized exclusively in the oocyte cytoplasm. No immunospecific protein was detected after injection of the DNA encoding mature chymosin. In terms of protein expression in oocytes, the Herpes simplex thymidine kinase (TK) promoter was up to sevenfold more effective than the simian virus 40 (SV40) early promoter, and equally as effective as the Moloney murine sarcoma virus long terminal repeat element. Where tested, protein expression in oocytes was much reduced if DNA sequences encoding the SV40 small t intron and its flanking sequences were present in the constructs. S1 nuclease mapping of transcripts produced after injection of DNAs containing the TK promoter indicated that the majority of transcripts initiated at, or within, two bases of the known cap site. However, minor transcripts initiating upstream from this site were observed and one (or more) of these transcripts was responsible for the synthesis of an ovalbumin polypeptide containing a 51 amino acid N-terminal extension. This extended protein remained in the oocyte cytosol. When ovalbumin cDNA was inserted into the vectors with opposite polarity to the viral promoter, expression in oocytes resulted in the predominant synthesis and secretion of a variant ovalbumin with a 21 amino acid N-terminal extension, although some full-length ovalbumin was also synthesized and secreted. S1 mapping revealed the presence, in these oocytes, of transcripts of predicted polarity initiating 118 bases upstream from the wild type ovalbumin initiator ATG, at a previously unreported SV40 promoter. No protein synthesis was detected after the injection of these reverse-orientation constructs into baby hamster kidney (BHK-21) cells.

Interactions of mouse immunoglobulin chains within Xenopus oocytes.

Oocytes of Xenopus laevis were used to study the fate of isolated MOPC 21 heavy (H) or light (L) immunoglobulin chains synthesized after microinjection of the encoding messenger RNAs. Tetrameric immunoglobulin molecules (H2L2) were formed within 24 hours after heavy and light chain mRNAs were injected separately into the same oocytes at diametrically opposed sites just beneath the oocyte plasma membrane; this indicates that diffusion of either the mRNAs or their membrane-sequestered products must occur throughout the cytoplasm. n nAs shown previously (Valle et al., 1981) each MOPC 21 chain becomes marooned within oocytes in the absence of the complementary chain. However, assembly and secretion of these chains were effected if the complementary chain was introduced via mRNA injection 24 hours later, indicating that the marooned immunoglobulin chains remain on the secretory route and occupy overlapping sites. n nFinally MOPC 21 heavy and light chain mRNAs and MPC 11 light chain mRNA were purified by filter hybridization to cloned complementary DNAs, and injected into oocytes. The secretion of MOPC 21 tetramer (H2L2) and the MPC 11 light chains occurred in the absence of other myeloma-specific proteins.

Synthesis of androgen-dependent secretory proteins by rat seminal vesicles

Androgenic steroids control the synthesis and secretion of several proteins by the seminal vesicles of the male rat. Prominent among them are 2 basic proteins, S and F, previously used as markers of androgen action. These proteins are not found among translation products of a wheat-germ protein-synthesising system supplied with mRNA from seminal vesicles of normal rats. Instead, higher molecular weight forms, S and F, are formed which are nonetheless related antigenically to S and F respectively. Injected into Xenopus Laevis oocytes, seminal vesicle mRNA does direct synthesis and secretion of polypeptides S and F. Partial peptide analysis confirms that S and F have extensive amino acid sequence homology with S and F respectively. We conclude that S and F are precursor forms of the secreted proteins and thus at least 2 abundant proteins of this male accessory tissue are secreted by a mechanism common to a wide number of secreted proteins.

Effect of anti-ER antibodies within the ER lumen of living cells

We describe the production and partial characterization of 12 monoclonal antibodies raised against a preparation of endoplasmic reticulum membranes obtained from Xenopus laevis liver. Four of the antibodies cross-react with liver melanocytes; two of the antibodies recognize extracellular antigens, whilst the remaining six recognize antigens present in hepatocytes. The concentrations of these latter antigens increase markedly in livers stimulated by estrogen. Western blotting analysis revealed that the six anti-hepatocyte monoclonal antibodies recognize at least five different antigens whose molecular weights are 14K, 18K, 19K, 43K, and 125K. The possible functional involvement of the various antigens in the secretory pathway was investigated using Xenopus oocytes as a surrogate secretory system. The mRNAs coding for the monoclonal antibodies were injected into oocytes and the resulting immunoglobulin chains were translated and assembled into active anti-ER antibodies inside the lumen of the ER. The effect on secretion was then observed. Our data indicate that the binding of antibodies to most antigens of the endoplasmic reticulum membrane may result in a blockage of secretion.

Expression of coronavirus E1 and rotavirus VP10 membrane proteins from synthetic RNA

Some viruses acquire their envelopes by budding through internal membranes of their host cell. We have expressed the cloned cDNA for glycoproteins from two such viruses, the E1 protein of coronavirus, which buds in the Golgi region, and VP10 protein of rotavirus, which assembles in the endoplasmic reticulum. Messenger RNA was prepared from both cDNAs by using SP6 polymerase and either translated in vitro or injected into cultured CV1 cells or Xenopus oocytes. In CV1 cells, the El protein was localised to the Golgi region and VP10 protein to the endoplasmic reticulum. In Xenopus oocytes, the E1 protein acquired post‐translational modifications indistinguishable from the sialylated, O‐linked sugars found on viral protein, while the VP10 protein acquired endoglycosidase‐H‐sensitive N‐linked sugars, consistent with their localisation to the Golgi complex and endoplasmic reticulum, respectively. Thus the two proteins provide models with which to study targeting to each of these intracellular compartments. When the RNAs were expressed in matured, meiotic oocytes, the VP10 protein was modified as before, but the E1 protein was processed to a much lesser extent than in interphase oocytes, consistent with a cessation of vesicular transport during cell division.