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.

Simian virus 40 large T antigen (T) can transform cultured cells, but the mechanisms by which it functions are not entirely understood. Several lines of evidence have suggested that the amino-terminal approximately 130 residues of T may be sufficient to confer the transforming capability. Oligonucleotide-directed mutagenesis was used to generate a series of deletion and substitution mutants within the amino-terminal 82 residues of T, the segment which is shared with simian virus 40 small t antigen (t). Results of stability and transformation assays of these mutants strongly suggest that the 1-to-82 region of T contains sequences which govern T transforming activity and affect in vivo stability. Instability and a defect in transforming activity could be separated from one another genetically. Thus, the 1-to-82 region appears to contain a specific region that contributes to the transforming function of the protein. This segment operates by means other than the simple binding of pRb and/or p107.

A series of replication-competent simian virus 40 (SV40) large T antigens with point and deletion mutations in the amino acid sequence between residues 105 and 115 were examined for the ability to immortalize primary cultures of mouse and rat cells. The results show that certain mutants, including one that deletes the entire region, are able to immortalize. However, consistent with previous data, the immortalized cells are not fully transformed, as judged by doubling time, sensitivity to concentrations of serum, and anchorage-independent growth. The region from 106 to 114 has structural features in common with a region involved in transformation by adenovirus E1a protein (J. Figge, T. Webster, T.F. Smith, and E. Paucha, J. Virol. 62:1814-1818, 1988) and influences the binding of the retinoblastoma gene product to large T (J.A. DeCaprio, J.W. Ludlow, J. Figge, J.-Y. Shew, C.-M. Huang, W.-H. Lee, E. Marsilio, E. Paucha, and D.M. Livingston, Cell 54:275-283, 1988). Together, these results imply that the sequence from 106 to 114 forms part of a domain that is essential for transformation of established cells, is dispensable for immortalization, and is not required for SV40 replication. The results also indicate that the ability of SV40 large T to immortalize primary cells is independent of its ability to bind to the retinoblastoma gene product.

Simian virus 40 large tumor (T) antigen stimulates transcription from the SV40 late promoter and some cellular genes. We report here the novel finding that purified T antigen preferentially stimulates transcription from the human heat shock protein 70 promoter in an in vitro transcription system. T antigen is thus capable of stimulating transcription by a process that does not require synthesis of other proteins and that may involve a direct interaction with preexisting cellular factors.

In addition to Rb and p53, a third cellular protein, p120 in monkey and p118 in human cells, forms a specific complex with SV40 large T antigen (T). p118120 is not a product of the Rb gene. As was shown with TRb complex formation, the interaction between T and p120 is dependent on the intact nature of a ten residue, transformation-controlling domain in T (residues 105–114). In mouse cells, a readily detectable protein of 115 kd was detected, which, like murine Rb, also forms a stable complex with T. Like p118120, p115 binding is also dependent on the intact nature of the 105–114 sequence. Given their similar size and T antigen binding sequence dependence, p115 and p118120 may be products of the same gene in different species. These results suggest that interactions between T and p115/118/120, as well as T and Rb, contribute to the SV40 transforming mechanism.

Extracts of monkey cells (CV-1P) synthesizing SV40 large T antigen (T) were immunoprecipitated with monoclonal antibodies to T or p110–114Rb, the product of the retinoblastoma susceptibility gene (Rb). While a family of p110–114Rb proteins can be detected in anti-Rb immunoprecipitates, only one member of this family, p110Rb, was found in anti-T precipitates of these extracts. Identical results were obtained with extracts of CV-1P cells which had been previously mixed in vitro with highly purified T. The p110–114Rb family is composed of two sets-p110Rb, an un- or under-phosphorylated species, and pp112–114Rb, a group of overtly phosphorylated proteins. Thus, T bound preferentially to the un- or underphosphorylated member of the family. In addition, T failed to alter the relative abundances of these species. These results suggest a model in which the growth suppression function of Rb is down modulated either by phosphorylation or T antigen binding.

Wild-type simian virus 40 large T antigen is very effective at blocking adipocyte differentiation in 3T3-F442A cells as assayed by triglyceride accumulation, induction of glycerophosphate dehydrogenase activity, and expression of mRNAs for glycerophosphate dehydrogenase, the adipocyte serine protease adipsin, and the putative lipid-binding protein adipocyte P2. Point mutants defective for either origin-specific DNA binding or transformation blocked differentiation as completely as wild type.

Regions containing similar elements of primary and predicted secondary structure were identified in simian virus 40 large T, adenovirus E1A, c-myc and v-myc proteins by a computer program with a set of highly specific, complex pattern descriptors. In all cases these regions were localized in domains of the respective proteins known to be required for transforming activity. We suggest that these apparently structurally similar regions may mediate a common biological function.

Monkey cells synthesizing SV40 large T antigen were lysed and the extracts immunoprecipitated with either monoclonal anti-T antibody or monoclonal antibody to p110–114, the product of the retinoblastoma susceptibility gene (Rb). T and p110–114 coprecipitated in each case, implying that the proteins are complexed with each other. Substitution and internal deletion mutants of T that contain structural alterations in a ten residue, transformation-controlling domain failed to complex with p110–114. In contrast, T mutants bearing structural changes outside of this domain bound to p110–114. These results are consistent with a model for transformation by SV40 which, at least in part, involves T/p110–114 complex formation and the perturbation of Rb protein and/or T function.

Fifty variant forms of simian virus 40 (SV40) large T antigen bearing point, multiple point, deletion, or termination mutations within a region of the protein thought to be involved in DNA binding were tested for their ability to bind to SV40 origin DNA. A number of the mutant large T species including some with point mutations were unable to bind, whereas many were wild type in this activity. The clustering of the mutations that are defective in origin DNA binding both reported here and by others suggests a DNA-binding domain on large T maps between residues 139 and approximately 220, with a particularly sensitive sequence between amino acids 147 and 166. The results indicate that the domain is involved in binding to both site I and site II on SV40 DNA, but it remains unclear whether it is responsible for binding to cellular DNA. Since all the mutants retain the ability to transform Rat-1 cells, we conclude that the ability of large T to bind to SV40 origin DNA is not a prerequisite for its transforming activity.

A short sequence of predominantly basic amino acids Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val from SV40 Large T is responsible for the normal nuclear location of the protein. Alteration of Lys-128 to each of six different residues other than Arg renders Large T cytoplasmic, whereas single amino acid changes in the surrounding region impair but do not prevent nuclear accumulation. When transposed to the amino terminus of cytoplasmic Large T species, or Escherichia coli beta-galactosidase or of chicken muscle pyruvate kinase, the sequence around Lys-128 of Large T is able to direct the recipient protein to the nucleus. This demonstrates that these amino acids can be sufficient for nuclear location and can act as a nuclear location signal. A computer search of over 2500 proteins reveals that some other nuclear proteins (for example, BK virus Large T, SV40 VP2 and adenovirus 72kDa DNA binding protein) contain very similar basic tracts, but so too do some presumed non-nuclear proteins (for example, poliovirus VP3). We suggest that the related sequence acts as the nuclear location signal in the other nuclear proteins but that the sequence does not function in all cases, perhaps because it is not accessible. A similar, but shorter or less basic sequence, was detected in a number of other nuclear proteins, for example, polyoma virus Large T, SV40 VP1 and several histones. However, such sequences were also found in many other proteins. Perhaps the shorter basic sequences can also act as nuclear location signals, but to be functional they need to be exposed (for example, at the amino terminus of the protein as in SV40 VP1) or to be present in multiple copies.

We have examined the large T encoded by an SV40 mutant, d10, which fails to localize to the nucleus. The DNA sequence of the mutant predicts the alteration of Lys 128‐‐‐‐Thr within the sequence 127 Lys Lys Lys Arg Lys 131 of large T. The results show that d10 large T is capable of binding to SV40 DNA, to cellular DNA and to the cellular phosphoprotein p53 as well as wild‐type large T. These data suggest that the cytoplasmic location of d10 large T is not due to an inability of the protein to be retained within the nucleus, but argues instead that the protein fails to reach the nucleus because it contains a defective nuclear location signal.

Antibodies were raised against six synthetic peptides corresponding to overlapping amino acid sequences (106 through 145) from a putative DNA binding domain in simian virus 40 (SV40) large-T antigens. All six antipeptide sera immunoprecipitated large-T from crude extracts of SV40-transformed cells, but the efficiency varied widely; in general, antibodies to the longer peptides produced the strongest anti-large-T activity. Antisera were purified by immunoaffinity chromatography on immobilized peptide. The purified antisera recognized only some forms of large-T; full-sized large-T from transformed cells, super-T from SV3T3 C120 cells, and 70,000-dalton T-antigen from Taq-BamHI cells were immunoprecipitated, whereas large-T from productively infected cells reacted irreproducibly, and the full-sized protein, synthesized in vitro or eluted from sodium dodecyl sulfate-containing gels, and the 33,000- and 22,000-dalton truncated large-Ts from Swiss SV3T3 and MES2006 cells, respectively, were not immunoprecipitated. This pattern of reactivity was explained when extracts were fractionated by sucrose density centrifugation, and it was found that only rapidly sedimenting forms of large-T were immunoprecipitated by the antipeptide sera; that is, large-T complexed with nonviral T antigen was detected, whereas lighter forms were not detected. Cascade immunoprecipitations did not support the view that this result was caused by the low affinity of the peptide antisera for large-T, and Western blotting experiments confirmed that the peptide antisera react directly with immobilized, monomeric large-T but not with nonviral T antigen. Immunoprecipitation assays to detect large-T:nonviral T antigen complexes bound specifically to fragments of SV40 DNA showed that under conditions of apparent antibody excess, DNA still bound to the complex.

A peptide Tyr.Arg.Asp.Leu.Lys.Leu corresponding to the carboxy-terminal six amino acids of small-t antigen predicted from the DNA sequence of SV40 was synthesised, coupled to bovine serum albumin and to ovalbumin and used to raise antibody in rabbits. The sera obtained immunoprecipitated [125I]peptide. It also recognised SV40 small-t that was synthesised in vitro from SV40 mRNA or extracted from SV40 infected monkey cells. The immunoprecipitation of small-t was inhibited by added peptide. To demonstrate that the determinant was present at the carboxy-terminal end of the molecule, truncated versions of small-t coded for by 0.54-0.59 deletion mutants were tested. dl 890 small-t, which contains an in-phase deletion removing nine amino acids but leaving the carboxy-terminal sequences intact, was recognised by the antipeptide serum. By contrast dl 885 small-t, which has an out-of-phase deletion leading to an altered carboxy terminus coded in an alternative reading frame, was not recognised. The data confirm the location and specificity of the determinant recognised on small-t by the antipeptide serum.

Two characteristics of cells transformed by the papovavirus SV40 are an increased cell-proliferation rate and the ability to cause tumors when infected into animals. Because these two phenomena are of fundamental importance in both biology and medicine, much effort is being concentrated on understanding how a relatively simple virus like SV40 is able to bring about such profound effects. Virally transformed cells contain viral DNA integrated into their chromosomes, they express viral RNA transcripts, and they contain viral proteins (for review, see Kelly and Nathans 1977). It is likely that at least some of the events occurring upon transformation are mediated by the viral proteins. However, before we can hope to understand the function of these viral proteins, we need a clear description of the various species present in transformed cells and of their interaction with hostcell components. Recent work has shown that in productively infected cells the early region...