Nucleotide Sequence of the Simian Virus 40 Hind‐K Restriction Fragment

Abstract

The restriction fragment Hind-K represents 4.2 ' %, of the genome of Simian virus 40 (SV40) and is located near the middle of the late region. Tts nucleotide sequence is reported here. It was mainly established by analysis of transcription products, synthesized by means of Esscherichiu coli RNA polymerase and nucleoside triphosphates, one of which was (~-~~P ) -l a b e l e d .Strand assignment was possible by hybridization of asymmetric, labeled transcripts of total SV40 DNA to filter-bound Hind-K fragment. Further information and unambiguous confirmation of the sequence was obtained by the use of direct DNA-sequencing methods. For this purpose the fragment was labeled at the 5' ends by means of polynucleotide kinase and [ Y -~~P I A T P and redigested with a suitable restriction enzyme. The separated products were then either partially digested with snake venom diesterase for analysis by the 'wandering spot' method or partially degraded with the base-specific reagents dimethylsulphate or hydrazine for direct sequence analysis on gel. The Hind-K sequence is 219 base pairs long. The message strand is particularly rich in adenosine (39 2) and purines. The nucleotide sequence can unambiguously be translated into an amino acid sequence and the N-terminal codon of the viral protein VP1 gene could be identified. The amino-terminal part of VP1 is rich in proline and lysine. The nucleotide sequence of Hind-K codes also for the carboxylterminal part of the viral protein VP2 and VP3 genes, which partly overlap the VP1 gene.In order to understand the organization of the genetic information in the oncogenic animal virus Simian virus 40 (SV40) in molecular detail, we have sequenced various parts of this genome. The viral DNA is a circular, double-stranded, supercoiled molecule containing approximately 5200 base pairs. It is cleaved by the HzndII+III restriction endonucleases into 13 fragments which were ordered [1,2]

Localization of the 5′ terminus of late SV40 mRNA

“…Digestion with ribonuclease A (Fig. 3c) clearly shows the products ApCp and ApUp (both of which are specific for the isomers present in the leader sequence) in equimolar amount with the product ApGp derived from the isomer UpCpCpAp Gp (n°121) present in the Hind II + III fragment K (10,23).…”

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

“…This open reading frame connects upstream (i.e. towards the section coding for the N terminus of the VP1 protein) with the open reading frame of Hind-F [21,22] and continues further through Hind-K [20] until it reaches the triplet for the N-terminal codon, dG-dC-dC, at the HaeIII restriction cleavage site close to the Hind-E/K junction [18]. Downstream (i.e.…”