Nucleotide sequence of the simian virus 40 Hind II + III restriction fragment J and the total amino acid sequence of the major structural protein VP1.

The HindII + III restriction fragment J (Hind-J) represents 4.58% of the simian virus 40 genome. The information present in Hind-J is expressed as part of the major, late 16-S messenger RNA, which codes for the structural protein VP1. The nucleotide sequence of the 240-base-pairs-long Hind fragment J has been determined by analysis of each oligonucleotide from both strands resulting from T1 or pancreatic RNase digestion of RNA transcribed from the DNA and from RNase digestion of ribo-substituted DNA. Large oligonucleotide blocks which could be constructed mainly on the basis of complementarity were subsequently ordered by partial chemical degradation of terminally labeled DNA. This direct DNA sequencing approach also completely confirmed the results obtained by both aforementioned RNase degradation methods. In the strand with the same polarity as the late mRNA, triplets corresponding to termination codons are present in two of the three reading frames. The one open reading frame connects in phase with the open reading frame of the neighboring HindII/ III fragments K, F and G, which have been published previously and which together with Hind-J span the total VP1 gene. Some features of the primary nucleotide sequence of this VP1 gene and the derived VP1 amino acid sequence are discussed.     

Nucleotide sequence of simian virus 40 DNA: structure of the middle segment of the HindII + III restriction fragment B (sixth part of the T antigen gene) and codon usage

We report here the nucleotide sequence of the simian virus 40 DNA region that lies between the EcoRII restriction endonuclease cleavage sites at map positions 0.214 and 0.281. The sequence was determined by partial chemical degradation of terminally labeled DNA fragments according to the procedure of Maxam and Gilbert. This region represents 6.7% of the SV40 genome and is located in the middle of HindII + III restriction fragment B. It is expressed as part of the early 19-S messenger RNA, which codes for the large-T antigen protein. Only one open reading frame for translation can be deduced from the message strand of the DNA and this reading frame connects in phase with the one of both neighboring fragments. This publication is the last in a series of papers about the T-antigen gene, and several properties of this gene and its product are discussed. The non-randomness of codon usage is similar to that previously discussed for the late part of the genome. Moreover, it appears that the choice of a third letter can be determined by the nature of the following codon; some codons which start with a pyrimidine are almost never preceded by an adenosine and some ANN-type codons are almost never preceded by a guanosine.     

Complete nucleotide sequence of the simian-virus 40 Hind-G fragment and localisation of the carboxyl terminus of the VP1 protein.

The restriction fragment Hind-G represents 7.0% of the simian virus 40 (SV40) genome. The information present in fragment Hind-G is expressed as part of the major, late 16-S messenger RNA. The complete nucleotide sequence of the fragment Hind-G has now been determined by application of the procedure of Maxam and Gilbert [Proc. Natl Acad. Sci. U.S.A. (1977) 74, 560-564]. It contains 369 nucleotide base pairs. On the basis of the termination code words in the strand with the same polarity as the late mRNA, two illegitimate reading frames can be defined. Therefore the third, open frame must code for the carboxyl terminal part of the VP1 protein. It terminates within fragment Hind-G with a TGA signal. This stop codon is followed by a non-translated region of the mRNA of about 83 nucleotides. The latter contains the sequence A-A-U-A-A-A, common to all other eukaryotic mRNA molecules so far studied. The Hind-G fragment also contains sequences which presumably play a role in the synthesis, processing and/or expression of early mRNA; these aspects are discussed in the following paper.     

Nucleotide sequence of the simian virus 40 HindII + III restriction fragment A (second part of the T antigen gene)

The nucleotide sequence of the second part of the simian virus 40 DNA HindII + III restriction fragment A is presented. The sequence extends from map position 0.533 to 0.424 and together with the first part of Hind-A [Volckaert et al. Proc. Natl Acad. Sci. U.S.A. 75, 2160--2164 (1978)] completes the total hind-A sequence, comprising 1169 base pairs. The second half of Hind-A includes the region corresponding to the second splicing boundary common to small tumor antigen (small-t) and large tumor antigen (large-T) mRNA and it contains coding information for an internal portion of large-T antigen. Two similar secondary structures of reasonable thermodynamic stability can be proposed for the nucleotide sequence of the pre-mRNA corresponding to the region reported here. Their possible relevance to the splicing of the SV40 early mRNAs is discussed. The deduced amino acid sequence is 188 residues long and contains a Lys-Lys-Lys-Arg-Lys-stretch which may be involved in the DNA binding capacity of large-T. A presumptive phosphorylation site is also present.     

Characterization of a gene encoding a 115 K super T antigen expressed by a SV40-transformed rat cell line.

It has been reported that SV40-transformed V 11 F 1 clone 1 subclone 7 rat cells (subclone 7) produce a super T antigen of 115,000 M. This super T antigen is entirely SV40 coded and is synthesized by translation of an elongated form of SV40 early mRNA (May, E., Kress, M. Daya-Grosjean, L., Monier, R. and May, P. (1981) J. Virol., 37, 24-35). The results reported here show that there is only one independent insertion of viral DNA in the cellular genome of subclone 7 cells. When DNA from subclone 7 cells was cleaved with Bam HI endonuclease two distinct SV40 sequence containing fragments were generated with sizes of 5 Kb and 10 Kb, respectively. Two recombinant cosmids were constructed by insertion of the 5 Kb and 10 Kb fragments, respectively, into cosmid pHC 79. Using restriction map analysis and nucleotide sequencing, we showed that the 5 Kb fragment actually contained the complete sequence of a gene encoding super T antigen. As compared to the normal SV40 early gene, the sequence of super T gene showed the following rearrangements: (i) The segment between nucleotides 4116 - 3544 was duplicated in a direct order and (ii) these two copies of 573 nucleotide sequence were separated by a 93 nucleotide tract which was a nearly perfect inverted repeat of the segment located between nucleotides 4868 and 4776 (nucleotide numbering used here = Weissmann number +17).     

Simian virus 40 and polyomavirus large tumor antigens have different requirements for high-affinity sequence-specific DNA binding.

By using a DNA fragment immunoassay, the binding of simian virus 40 (SV40) and polyomavirus (Py) large tumor (T) antigens to regulatory regions at both viral origins of replication was examined. Although both Py T antigen and SV40 T antigen bind to multiple discrete regions on their proper origins and the reciprocal origin, several striking differences were observed. Py T antigen bound efficiently to three regions on Py DNA centered around an MboII site at nucleotide 45 (region A), a BglI site at nucleotide 92 (region B), and another MboII site at nucleotide 132 (region C). Region A is adjacent to the viral replication origin, and region C coincides with the major early mRNA cap site. Weak binding by Py T antigen to the origin palindrome centered at nucleotide 3 also was observed. SV40 T antigen binds strongly to Py regions A and B but only weakly to region C. This weak binding on region C was surprising because this region contains four tandem repeats of GPuGGC, the canonical pentanucleotide sequence thought to be involved in specific binding by T antigens. On SV40 DNA, SV40 T antigen displayed its characteristic hierarchy of affinities, binding most efficiently to site 1 and less efficiently to site 2. Binding to site 3 was undetectable under these conditions. In contrast, Py T antigen, despite an overall relative reduction of affinity for SV40 DNA, binds equally to fragments containing each of the three SV40 binding sites. Py T antigen, but not SV40 T antigen, also bound specifically to a region of human Alu DNA which bears a remarkable homology to SV40 site 1. However, both tumor antigens fail to precipitate DNA from the same region which has two direct repeats of GAGGC. These results indicate that despite similarities in protein structure and DNA sequence, requirements of the two T antigens for pentanucleotide configuration and neighboring sequence environment are different.     

Genomic structure of human polyoma virus JC: nucleotide sequence of the region containing replication origin and small-T-antigen gene

The nucleotide sequence of the region of human polyoma virus JC DNA between 0.5 and 0.7 map units from a unique EcoRI cleavage site was determined and compared with those of the corresponding regions of another human polyoma virus, BK, and simian virus 40 DNAs. Within this region consisting of 945 base pairs, we located the origin of DNA replication near 0.7 map units, the entire coding region for small T antigen, and the splice junctions for large-T-antigen mRNA. The deduced amino acid sequences for small T antigen and the part of large T antigen markedly resembled those of polyoma virus BK and simian virus 40. The results strongly suggest that polyoma virus JC has the same organization of early genome as polyoma virus BK and simian virus 40 on the physical map, with the EcoRI site as a reference point.