Differentiation (1987) 34: 68-78 The legend to Fig. 7a, b contained three printing errors (p instead of 4). The correct reading of lanes 1-5 is as follows: Lane 1, lpter-lp34; lane 2, lp34-lqter; lane 3, lp34-lqter; lane 4 , lpter-lp36.13; lane 5, lp36.13-lqter;

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Messenger RNAs from one kind of secretory cell can be introduced into the cytoplasm of another: the heterologous proteins formed by the recipient cell are usually processed and topologically segregated in the manner characteristic of the donor cell. Xenopus oocytes injected with honey-bee venom gland RNA provide some support for this generalization, but also reveal important exceptions to it. Thus, the frog cell makes a small polypeptide whose partial sequence matches perfectly that of insect promelittin, except that the product formed in oocytes ends at the C terminus with a glycine as opposed to a glutamine amide residue. N-terminal heterogeneity is seen in protoxin made in oocytes and venom gland cells, and species shorter by two residues are seen in both tissues. We suggest that the oocyte contains a dipeptidylpeptidase. Promelittin made in oocytes is barely detectable in the cytosol but is found associated with a vesicle fraction which also contains some newly synthesized endogenous oocyte proteins. The association with vesicles is long-lasting ; thus promelittin is retained slightly more efficiently than sequestered oocyte proteins, and an incubation period of about two weeks is required to reduce by half the amount of these endogenous vesicle proteins. Thus neither promelittin nor any products derived from it are secreted rapidly. Gel analysis fails to reveal promelittin in the medium surrounding the oocyte, although traces can be detected by assaying for a characteristic heptapeptide. Such small amounts could result from slow secretion or leakage. Melittin could not be detected by gel analysis or peptide assay. The retention of the honey-bee protein within the frog cell is discussed in terms of the specificity of the processing systems and secretory pathways of venom gland cells and oocytes. We suggest that whilst some export mechanisms function efficiently in a wide variety of cells, others do not, and may even be restricted to specific cell types.Proteins characteristic of one kind of cell can be synthesized in the cytoplasm of another [I -41 : their fate can reveal the nature and specificity of the posttranslational machinery [5]. Moreover, the factors affecting the subcellular localization of newly made proteins can be studied by fractionation of cells making such heterologous proteins. The microinjection of secretory protein messengers into Xenopus oocytes shows the presence of a secretory pathway [6-81, which is selective but appears to lack both species and cell-type specificity. Detailed studies require knowledge of the precise structure of both the newly made secretory protein and its processed forms. Honey-bee prepromelittin meets these requirements for the amino acid sequence has been determined [9] and, for many parts of the molecule, structure has been correlated with function. Thus four different regions, starting from the amino end, have been identified in the primary translation product; as shown in Fig. 1 there is a pre or signal sequence of 21 residues, a pro part comprised of 22 amino ...

We have investigated developmental expression of the gene Egr-1, which encodes a protein containing three zinc fingers. Egr-1 like c-fos is a serum inducible, early response gene, which is co-induced with c-fos in a variety of quite different situations. A single 3.7-kb RNA was detected throughout fetal mouse development, which increased in absolute levels in total fetal RNA from 9.5 to 12.5 days post coitum (p.c.). In situ hybridization to 14.5- and 17.5-day p.c. fetal tissues demonstrated Egr-1 accumulation at several specific sites. These included mesenchymal components of the developing tooth germs and salivary and nasal glands; an ectodermally derived component of the whisker pad and developing muscle, cartilage, and bone. Expression of Egr-1 in cartilage and bone showed a strikingly similar expression to previously published reports of c-fos in these tissues. High levels of Egr-1 RNA was observed at the perichondrial interface of opposing cartilaginous elements and in interstitial cells that lie in between. Bone expression was observed in membranous bone of the head, alveolar bone around the tooth germs, and at periosteal and endochondral ossification sites in the limb bones. Our data support the idea that Egr-1 and c-fos may be coregulated in vivo and together may regulate normal development of the skeleton.

The pattern of protein synthesis in 8-, 9- and 10-day post coitum (p.c.) mouse embryos was examined by 2-D gel electrophoresis of [35S]methionine-labelled proteins. Of the 600–800 polypeptides detected only one, a 14 X 10(3) Mr (14K) protein, was found to accumulate over this period. To isolate cDNA clones that potentially encode this protein, 32P-labelled cDNA was synthesized from 9 and 10 days p.c. embryo poly(A) +RNA, and used for the differential screening of an 8.5-day p.c. mouse embryo cDNA library cloned in lambda gt10. Six clones that hybridized strongly to the 10-day probe were purified and their inserts subcloned into plasmid vectors. Cross hybridization and restriction mapping of these inserts indicate that they fall into four distinct groups. Each of these hybridize with transcripts of approximately 600 nucleotides, which accumulate in the embryo from 9 to 10 days p.c. Expression was barely detectable in adult tissues and restricted to liver and spleen. Expression of one of these clones, 10.1, was examined by in situ hybridization of 35S-labelled RNA probes to 8.5-12.5 day p.c. embryo sections. Strong hybridization was observed in yolk sac blood islands, fetal liver and embryonic erythrocytes, suggesting that 10.1A encodes an erythrocyte-specific protein. DNA sequence analysis indicates that the four classes of cDNA were derived from transcripts of the alpha 1, zeta, beta h1 and epsilon globin genes. Labelling of 10-day p.c. erythrocyte proteins with [35S]methionine, followed by 2-D gel electrophoresis, clearly demonstrates that the most abundant polypeptide migrates to the same position as the 14K protein which accumulates from 8 to 10 days p.c. Thus the only abundant transcripts and corresponding proteins that change over a period of profound morphogenetic change correspond to globins of the newly established blood system.

Combining messenger RNA from one kind of secretory cell with the cytoplasm of another such cell can reveal the nature and specificity of protein export mechanisms. We show that messenger RNAs from secretory cells of chickens, rats, mice, frogs, guinea-pigs, locusts and barley plants, when injected into Xenopus oocytes, direct the synthesis and export of proteins. Chicken ovalbumin, Xenopus albumin, mouse thyroid-stimulating hormone, locust vitellin and guinea-pig milk proteins were identified using specific antibodies, whilst chicken lysozyme and ovomucoid, rat albumin, Xenopus vitellogenin and rat seminal vesicle basic proteins were identified provisionally from their molecular weights. Certain endogenous proteins are sequestered and secreted although most oocyte proteins are not exported. Similarly the major polyoma viral protein and the simian virus 40 and polyoma tumour antigens are retained within the oocyte. Radioactive proteins exported by oocytes programmed with chicken oviduct or Xenopus liver RNA are not re-exported in detectable amounts when injected into fresh oocytes, nor is there secretion of chicken oviduct or guinea-pig mammary gland primary translation products prepared using wheat germ extracts. Thus the export of secretory proteins from oocytes cannot be explained by leakage and may require a cotranslational event. The secretory system of the oocyte is neither cell-type nor species-specific yet is highly selective. We suggest that the oocyte can be used as a general surrogate system for the study of gene expression, from transcription through translation to the final subcellular or extracellular destination of the processed protein.The introduction of messenger RNA from one kind of cell into the cytoplasm of another reveals the general nature of the translational machinery [l --41. The injection of secretory protein messengers into Xenopus oocytes and other cells [ 5 ] suggests that the transfer of newly made polypeptides across the endoplasmic reticulum [6,7] lacks both cell-type and species specificity. Recently it has been shown [S] that oocytes of Xenopus luevis will export certain secretory proteins, such as caseins, whose synthesis is directed by heterologous mRNA. This process is highly selective since a non-secretory protein, rabbit haemoglobin, is not exported. In this paper we extend these observations to a range of proteins, secretory and non-secretory, whose mRNAs were derived from a variety of species. We show that whilst secretion is highly selective, it lacks cell-type or species specificity..~ Abhreviutions. T-antigen, large tumour antigen; t-antigen, small tumour antigen.The demonstration of specific export and specific intracellular localization of secretory proteins recommends the Xenopus oocyte as a general system for studying the secretory pathway [9]. MATERIALS AND METHODS Ooc.yte IncubationOocytes were injected with messenger RNA [l] and cultured [l] in modified Barth x medium [lo] (containing additional antibiotics : penicillin 100 units/ml, streptomycin 100 units...

The cytoplasm of the Xenopus oocyte can be altered by the microinjection of proteins and the regulatory responses to such perturbations can then be studied. We have investigated proteolytic systems within the oocyte which may be involved in the maintenance of the integrity of the different subcellular compartments. Thus primary translation products, made in the wheat germ system under the direction of frog liver, chicken oviduct, rat liver rapidly sedimenting endoplasmic reticulum, rat seminal vesicle, guinea pig mammary gland or honey been venom gland RNA, were injected into oocytes. Their stability in the frog cell cytosol was in general low compared to that of their processed counterparts. The latter were usually obtained by collecting the heterologous proteins exported by RNAinjected oocytes. Electrophoretic analysis of oocytes injected with particular primary and processed polypeptides permitted measurement of the stabilities of proteins differing only by the presence or absence of a detachable signal sequence, or by the presence of a specific secondary modification. The effect of the latter on protein stability appears slight. However, the presence of a detachable signal sequence destabilizes those miscompartmentalized secretory proteins which are otherwise stable. Indeed all other results are consistent with this concept for they show that primary translation products are in general much less and are never more stable than their processed counterparts. Thus we provide evidence that errors of compartmentation can be corrected in living cells and that this process is often facilitated by the properties conferred on a protein by a detachable signal sequence. WI. MATERIALS AND METHODSRadioactive proteins were injected into oocytes and their stabilities measured [7] : thus wheat germ extracts and oocyte incubation media were made 10 mM in methionine, were Abbreviation. SDS, sodium dodecyl sulphate.frozen, thawed and, without further treatment (thereby avoiding further possible denaturation artefacts), the protein solutions were introduced into the oocyte cytosol. In most experiments, a given protein solution was only thawed once: and, in certain experiments designed to control against denaturation artefacts, samples were injected without prior freezing. The recipient cells were both preincubated (4 h) and incubated in medium containing 15 mM methionine, thus preventing reincorporation of radioactive amino acid. The same micropipette, calibrated to contain about 350nl of solution, was used in a given series of stability measurements. Two pipettefuls of solution, yielding about 14 injected oocytes, were used for each time point. The batches of injected oocytes were incubated for up to 24h: at various times oocytes and their surrounding media were measured for their content of total and acid-insoluble radioactivity and then analyzed on SDS/polyacrylamide gels. Batches of frog cells showing leakage [l 11 were discarded. Radioactive polypeptides resolved on SDS/polyacrylamide gels were revealed by autoradiography o...