Regions of the polyoma genome coding for T antigens

Hunter, Tony; Hutchinson, Mary Anne; Eckhart, Walter; Friedmann, Theodore; Esty, A; LaPorte, Patricia; Deininger, Prescott L.

doi:10.1093/nar/7.8.2275

Cited by 20 publications

(10 citation statements)

References 20 publications

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“…These are the 140-k and 170-K proteins (Pelham, 1979) and the 29-K and 16-K proteins described here. However, in TMV a larger number of gene products may be produced by translational initiation at sites within the three main open reading frames (Goelet and Kam, 1982;Hunter et al, 1983;Sulzinski et al, 1985) from specific subgenomic RNAs. The Northern blot analysis ( Figure 3) suggests that this strategy is not used with TRV, as multiple discrete subgenomic length RNA species are not detected.…”

Section: Rna1mentioning

confidence: 99%

The organisation and interviral homologies of genes at the 3′ end of tobacco rattle virus RNA1

Boccara¹,

Hamilton²,

Baulcombe³

1986

The EMBO Journal

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The RNA1 of tobacco rattle virus (TRV) has been cloned as cDNA and the nucleotide sequence determined of 2 kb from the 3′‐terminal region. The sequence contains three long open reading frames. One of these starts 5′ of the cDNA and probably corresponds to the carboxy‐terminal sequence of a 170‐K protein encoded on RNA1. The deduced protein sequence from this reading frame shows homology with the putative replicases of tobacco mosaic virus (TMV) and tricornaviruses. The location of the second open reading frame, which encodes a 29‐K polypeptide, was shown by Northern blot analysis to coincide with a 1.6‐kb subgenomic RNA. The validity of this reading frame was confirmed by showing that the cDNA extending over this region could be transcribed and translated in vitro to produce a polypeptide of the predicted size which co‐migrates in electrophoresis with a translation product of authentic viral RNA. The sequence of this 29‐K polypeptide showed homology with two regions in the 30‐K protein of TMV. This homology includes positions in the TMV 30‐K protein where mutations have been identified which affect the transport of virus between cells. The third open reading frame encodes a potential 16‐K protein and was shown by Northern blot hybridisation to be contained within the region of a 0.7‐kb subgenomic RNA which is found in cellular RNA of infected cells but not virus particles. The many similarities between TRV and TMV in viral morphology, gene organisation and sequence suggest that these two viral groups may share a common viral ancestor.

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Section: Rna1mentioning

confidence: 99%

The organisation and interviral homologies of genes at the 3′ end of tobacco rattle virus RNA1

Boccara¹,

Hamilton²,

Baulcombe³

1986

The EMBO Journal

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“…p30 is only one of a family of C-coterminal proteins made in vitro from the 30-K ORF on TMV RNA; at least two smaller proteins exist whose N termini map at methionine residues within the 30-K ORF (Hunter et al, 1983). In order to map the unique tryptic peptides more precisely, we prepared 14C-labelled tryptic peptide maps of a smaller member of the family, p19 (formerly known as 23 K from its apparent SDS gel mobility), from all three viruses .…”

Section: Screening For Mutations Using Tryptic Peptide Mappingmentioning

confidence: 99%

“…A, B, X, X', X", Y and Y' are peptides mentioned in the text. U, p19 was a product of RNA extracted from viral particles partly stripped with alkali (Hunter et al, 1983), all the other proteins were products of unfractionated viral RNA.…”

Section: Screening For Mutations Using Tryptic Peptide Mappingmentioning

confidence: 99%

“…Virus was extracted from uninoculated upper leaves. Local lesion assay and temperature shift Two or three pairs of opposite half leaves of greenhouse grown N. tabacum cv Xanthi nc (6-9 weeks old) were inoculated with purified virus (I-30 In vitro translation and peptide mapping Reticulocyte lysate conditions and peptide mapping protocol were described previously (Pelham and Jackson, 1976;Hunter et al, 1983 and references therein). The [14C]amino acid mixture (NEN catalogue no.…”

Section: Virusesmentioning

confidence: 99%

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Point mutation in the 30-K open reading frame of TMV implicated in temperature-sensitive assembly and local lesion spreading of mutant Ni 2519

Zimmern

Hunter

1983

The EMBO Journal

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Tobacco mosaic virus mutant Ni 2519 forms local lesions on tobacco cultivars carrying the N gene which, unlike wild‐type lesions, do not enlarge at elevated temperature. This may reflect temperature sensitivity of a viral gene product required for cell to cell spreading of infectivity. Ni 2519 also carries an unselected cis‐dominant lesion in viral assembly. Peptide mapping of in vitro translation products of Ni 2519 RNA reveals at least one, and possibly two changes in p30 and p19, two products of the 30‐K open reading frame, compared with its parental strain A14. An A to G transition at position 5332 in Ni 2519 RNA accounts for the altered mobility of the major variable peptide. The corresponding A14 peptide itself differs from the wild‐type due to another A to G transition at residue 5329. These residues are close to the viral assembly origin. A revertant virus population which could assemble at the restrictive temperature regained the wild‐type sequence in place of the point mutation specific to Ni 2519 at position 5332, and formed wild‐type local lesions as efficiently as the parental strain. This result implicates mutation of residue 5332 in the temperature sensitivity of viral assembly (by altering the structure of the RNA close to the assembly origin) and/ or local lesion spreading (via a radical Arg to Gly substitution in p30 or its derivatives). The mutation occurs in a position where the predicted amino acid sequence shows homology with a group of proteins encoded by yeast mitochondrial introns.

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“…Spot 1 is the acetylated N-terminal peptide of the small T antigen (2). Peptide NA1 migrated with a net positive charge, as would be predicted for a nonacetylated form of the Nterminal peptide, methionine-aspartic acid-arginine, produced by the bacteria.…”

mentioning

confidence: 73%

Syntheses and stabilities of proteins related to the polyoma small T antigen in Escherichia coli.

Horwich¹,

Koop²,

Eckhart³

1982

Mol. Cell. Biol.

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We compared the syntheses and turnovers of two proteins related to the polyoma small T antigen synthesized in Escherichia coli from plasmids containing polyoma genomic segments joined to lac control elements. A protein with an authentic polyoma N terminus was more unstable than a protein with N-terminal amino acids derived from 0-galactosidase. Both were more unstable than most bacterial proteins.The polyoma and simian virus 40 (SV40) genomes encode early proteins (T antigens) which are involved in virus replication and neoplastic cell transformation (see reference 6 for review). Polyoma encodes three T antigens (large, medium [or middle], and small); SV40 encodes two (large and small). It would be useful to be able to synthesize the T antigens in large amounts to facilitate their purification and characterization. Approaches to doing this are being developed both in animal cells and in bacteria. The SV40 large T antigen has been expressed at high levels in mamalian cells, under the control of an adenovirus late promoter (5). The SV40 small T antigen has been expressed at high levels in Escherichia coli, under the control of a lac promoter (4).We previously described the synthesis in E. coli of a protein related to the polyoma small T antigen (1). This protein is expressed by a plasmid containing a segment of the polyoma genome joined to a lac control element in such a way that a "fused" protein results. The fused protein is initiated in 0-galactosidase sequences in the lac control element and is continued into the polyoma small-T-antigen coding region in the correct frame for translation. We have now constructed additional plasmids containing another lac control element which permitted the synthesis of a nonfused protein, initiated at the small-T-antigen initiation codon. We describe here the syntheses of the two proteins and a comparison of their stabilities in E. coli.We joined the coding region for the polyoma small T antigen to a lac operon control element in such a way that translation would begin at the initiation codon for the small T antigen. We used the general approach described by Roberts et al. (3), seeking to place the ribosome binding sequence of the lac control element at an optimum distance from the initiation codon. The optimum distance is not precisely predictable but is often seven to nine nucleotides. Therefore, we constructed a group of plasmids having the ribosome binding site at various distances from the small-T-antigen initiation codon and tested their abilities to produce a protein related to the small T antigen.The procedure is summarized in Fig. 1. We cleaved a plasmid, pgt-6, containing the small-Tantigen coding region, at an EcoPJ restriction enzyme site 16 nucleotides upstream from the initiation codon. We digested the plasmid DNA briefly with exonuclease III and then with S1 to produce molecules having ends at various distances less than 16 nucleotides from the initiation codon, as shown in Fig. 2. We then inserted a lac control fragment into the digested plasmid molecules. The...

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Regions of the polyoma genome coding for T antigens

Cited by 20 publications

References 20 publications

The organisation and interviral homologies of genes at the 3′ end of tobacco rattle virus RNA1

The organisation and interviral homologies of genes at the 3′ end of tobacco rattle virus RNA1

Point mutation in the 30-K open reading frame of TMV implicated in temperature-sensitive assembly and local lesion spreading of mutant Ni 2519

Syntheses and stabilities of proteins related to the polyoma small T antigen in Escherichia coli.

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