Stanley T. Bayley

A region in the C-terminus of adenovirus 2/5 E1a protein is required for association with a cellular phosphoprotein and important for the negative modulation of T24-ras mediated transformation, tumorigenesis and metastasis.

We have examined a series of small deletion mutants within exon 2 of the adenovirus 2/5 E1A oncogene product, the 243R protein, for immortalization, ras cooperative transformation, tumorigenesis and metastasis. Compared with wild‐type 243R, various deletion mutants located between residues 193 and 243 cooperated more efficiently with ras to induce large transformed foci of less adherent cells that were tumorigenic and metastatic. However, the greatest enhancement of transformation (comparable to that obtained with a deletion of the C‐terminal 67 amino acids) was observed with a mutant carrying a deletion of residues 225–238. This mutant was also more defective in immortalization. These results suggest that this 14 amino acid region may contain a function that is important for immortalization and negative modulation of tumorigenesis and metastasis. To identify cellular proteins that may associate with the exon 2‐coded region of E1A (C‐terminal half) and modulate its transformation potential, we constructed a chimeric gene coding for the C‐terminal 68 amino acids of E1a fused to bacterial glutathione‐S‐transferase (GST). This fusion protein was used to purify cellular proteins that bind to the C‐terminal region of E1a. A 48 kDa cellular protein doublet (designated CtBP) was found to bind specifically to the GST‐E1a C‐terminal fusion protein as well as to bacterially expressed full‐length E1a (243R) protein. It also co‐immunoprecipitated specifically with E1a. Analysis of a panel of GST‐E1a C‐terminal mutant proteins indicates that residues 225–238 are required for the association of E1a and CtBP, suggesting a correlation between the association of CtBP and the immortalization and transformation modulating activities of exon 2. CtBP is a phosphoprotein and the level of phosphorylation of CtBP appears to be regulated during the cell cycle, suggesting that it may play an important role during cellular proliferation.

Mapping of cellular protein-binding sites on the products of early-region 1A of human adenovirus type 5.

The binding sites for the 300-, 107-, and 105-kilodalton cellular proteins which associate with human adenovirus type 5 E1A products were studied with E1A deletion mutants. All appeared to bind to the amino-terminal half of E1A products in regions necessary for oncogenic transformation. These results suggest that these cellular species may be important for the biological activity of E1A products.

Sequences in E1A proteins of human adenovirus 5 required for cell transformation, repression of a transcriptional enhancer, and induction of proliferating cell nuclear antigen

A range of deletion and other mutants in the coding region of the E1A gene of Ad5 has been assayed for transformation of baby rat kidney (BRK) cells in cooperation with ras, repression of the SV40 enhancer, and induction of proliferating cell nuclear antigen (PCNA). Transformation efficiency was drastically reduced by deletion of residues 4-25, 36-60, or 111-138 in exon 1 of the 289 residue (289R) and 243R E1A proteins. Deletion of other residues in exon 1 had little effect. With mutants in the region unique to the 289R protein, and in exon 2, the only effect on transformation seemed to be an increased tendency of mutant transformants, compared to wt, to migrate to form secondary foci. Repression assays, performed with E1A plasmids producing only the 243R protein, showed that deletion of residues 4-25 or 36-60 inhibited repression completely. Deletion of residues 128-138 reduced repression, but deletions elsewhere in exon 1 had little effect. Deletion of residues 188-204 in exon 2 reduced repression slightly, and deletion of all of exon 2 reduced it to about one-half. It is concluded that for transformation, there are two functional domains in E1A proteins, both in exon 1, both involved in binding different cellular proteins, and both probably concerned with different transforming functions. One of these domains, involving residues 4-25 and 36-60, also functions in repression, but the role of the second in repression is much less critical. All of the deletion mutants in exon 1 induced PCNA synthesis in BRK cells. This result, together with previously published work, suggests that the active site for PCNA induction either involves residues 61-69 or 82-85 in exon 1, which have not been deleted, or it does not depend on any single limited region of the E1A proteins.

Recent Developments in the Molecular Biology of Extremely Halophilic Bacteria

(1978). Recent Developments in the Molecular Biology of Extremely Halophilic Bacteria. CRC Critical Reviews in Microbiology: Vol. 6, No. 2, pp. 151-206.

Effects of Ad5 E1A mutant viruses on the cell cycle in relation to the binding of cellular proteins including the retinoblastoma protein and cyclin A

We have examined the ability of Ad5 E1A 12S viruses with deletions in E1A exon 1 to induce quiescent baby rat kidney cells to progress through the cell cycle and to undergo mitosis. Measurements of mitotic index and analyses by fluorescence activated cell sorting were correlated with the abilities of the mutant E1A proteins to bind to cellular proteins. All the mutants induced cells to leave G0/G1 and enter S phase, but two groups were defective at inducing mitosis, and cells infected with them appeared to be blocked between the S and M phases. The first group of mutants, with deletions in the regions of residues 4-25 and 30-60, bound p300 poorly or not at all and gave reduced numbers of mitoses. The second group, with deletions between residues 111 and 138 in CR2, failed to bind pRb and were completely defective at inducing mitosis. In this group, mutants lacking residues between 124 and 138 bound p107 and cyclin A at much reduced levels and induced cells to overreplicate their DNA. The site in E1A required to bind cyclin A extends from residue 124 to at least 127. Cyclin A binds to a 107-kDa cellular protein, which by peptide analysis appears identical to p107.

Transformation by human adenoviruses

When, approximately 10 years ago, it was shown that the functions essential for cell transformation were localized in a small region of the adenovirus genome, a DNA segment which at that time was thought to be capable of encoding two or three average-sized proteins at most, it seemed reasonable to hope that an understanding of the mechanisms by which adenoviruses transform cells might be quickly achieved. While such optimism might be forgiven, it was quite clearly naive in the extreme. As a consequence of mRNA splicing and the use of overlapping reading frames the number of proteins encoded within E1 is 2-3-times greater than would have been predicted a decade ago, and post-translational modifications may add another dimension of complexity. In fact it has taken nearly all of the past decade just to identify the proteins encoded in E1 and to characterize them in the most rudimentary way. However, we have now entered a period in which new information is accumulating at an extremely rapid rate as a result of several major technical and fundamental advances. Chief among these are the use of recombinant DNA techniques, particularly site-directed mutagenesis, which combined with methods for introducing mutations made in cloned sequences back into infectious virus, clearly represents a powerful approach to studying the functions of transforming proteins. In addition, the ability to express transforming proteins in bacteria and to produce large amounts of highly purified proteins which previously were only just detectable in infected and transformed cells is a major breakthrough. Advances in immunological techniques, particularly the development of monoclonal antibodies and antisera against synthetic peptides, have enormously simplified the task of detecting and characterizing E1 proteins. Finally, recent results suggesting that adenovirus transforming proteins may be functionally and structurally similar to other oncogenes brings a new perspective to the study of oncogenic transformation. Have all the proteins involved in transformation by adenoviruses been identified? It seems probable that all those virally coded proteins which play a major role are now known but of course minor players in the cast could still be waiting in the wings. We have pointed out that viral functions encoded outside region E1 may have some importance at least in initiation of transformation by virions and have speculated on the possibility that one or more of these may be involved in the integration of viral DNA into the host cell chromosome.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of deletion and point mutants spanning the coding region of the adenovirus 5 E1A gene to define a domain that is essential for transcriptional activation

To help in identifying functional domains within Ad5 E1A proteins, we have constructed a series of mutants that create deletions throughout these products. We have also produced several mis-sense point mutations in the unique 13 S mRNA region. These mutated E1A regions have been tested in plasmid form for their ability to activate transcription of an E3-promoted CAT gene. From the results, a major domain for transactivation has been identified. This begins between residues 138 and 147, ends between residues 188 and 204, and encompasses the unique 13 S region. This domain is sensitive to mis-sense mutations. Transactivation was unaffected by small deletions in the N-terminal half of E1A proteins between residues 4 and 138, but was destroyed when this whole region was deleted. The C-terminal 71 residues may affect transactivation, but the results with the mutant in which this region was deleted were variable. The results obtained with these mutants are discussed in relation to the transactivation obtained by J. W. Lillie et al. [(1987). Cell 50, 1091-1100] with a synthetic peptide similar to the domain described here.

Tumor antigens of human ad5 in transformed cells and in cells infected with transformation-defective host-range mutants

We have studied the polypeptides associated with the expression of the transforming region of the Ad5 genome by immunoprecipitating antigens (using the double antibody and protein A-Sepharose techniques) from cells infected with wild-type (wt) Ad5 or transformation-defective host range (hr) mutants and from cells transformed by Ad5. Three different antisera were used: P antiserum specific for early viral products (Russell et al., 1967) and two different hamster tumor antisera. Immunoprecipitation of antigens from wt-infected KB cells followed by SDS-polyacrylamide gel electrophoresis of precipitated proteins revealed that a major polypeptide having a molecular weight of approximately 58,000 was detected with all three antisera and with both the double antibody and the protein A-Sepharose techniques, while P antiserum also precipitated polypeptides of molecular weights 72,000, 67,000 and 44,000, which probably represent the DNA binding protein and related polypeptides, respectively. With the double antibody technique, in addition to the proteins mentioned above, P antiserum and the hamster tumor antisera precipitated a 10,500 dalton polypeptide which was not detected when the protein A-Sepharose procedure was used. Using either the double antibody or the protein A-Sepharose technique, we found that hr mutants from complementation group II failed to induce the synthesis of the 58,000 dalton protein, whereas mutants from complementation group I produced normal or near normal amounts. Using the double antibody technique, we found that the 10,500 dalton protein was absent or made in reduced amounts by group I mutants. A 58,000 dalton protein was detected in a number of different Ad5-transformed cell lines, including the 293 human line, the 14b hamster line and several transformed rat cell lines. This observation and the fact that transformation negative group II mutants fail to induce the synthesis of a 58,000 dalton polypeptide suggest that this protein is one of the Ad5-specific products necessary for cell transformation.

Methionine transfer RNAs from the extreme halophile, Halobacterium cutirubrum

Abstract 1. 1. Formylation of methionyl-tRNA could not be demonstrated in Halobacterium cutirubrum, either in vitro or in vivo. 2. 2. Chromatography of [14C]methionyl-tRNAMet on BD-cellulose columns resolved two peaks. 3. 3. One of these chromatographically distinct species was capable of being formylated by the Escherichia coli methionyl-tRNA transformylase.

Synthesis of DNA, late polypeptides, and infectious virus by host-range mutants of adenovirus 5 in nonpermissive cells.

Transformation of cells by human adenoviruses appears to be brought about by “early” sequences located close to the lefthand end of the viral DNA molecule and covering approximately 7 to 8% of the genome. Several lines of evidence support this conclusion. First, Gallimore et al. (I) have shown that the left-hand 12 to 14% of the Ad 2 genome is always present in Ad 2transformed rat cells. Second, Flint et al. (2, 3) have shown that sequences at the left-hand end corresponding to about 7% of the viral genome are transcribed in transformed cells. Finally, studies on the biological activity of purified viral DNA have shown that only the left-hand 7 to 8% of the Ad 5 genome is necessary to transform rat kidney cells (4, 5). Transformation by human adenoviruses is not restricted to rodent cells. A line of Ad 5-transformed human embryonic kidney cells, line 293, has been established which shares many of the properties of transformed rat cells (6). The 293 cells contain the extreme left-hand 12.5% of the Ad 5 genome plus 10% of the right-hand end (R. Weinmann, personal communication) and transcribe sequences at the left-hand end ( 6). The 293 cells also contain viral products detected as T antigen(s) by indirect immunofluorescence using tumor antisera (5, 6). As an aid to elucidating the way adenoviruses bring about transformation, Harrison et al. ( 7) have isolated host-range mutants of Ad 5 which plaque on 293 cells but