A. Van de Voorde

Overlapping of the VP2-VP3 gene and the VP1 gene in the SV40 genome

The nucleotide sequence of the SV40 Hind E fragment has been determined mainly by the partial chemical degradation procedure of Maxam and Gilbert (1977). The sequence of the strand with the same polarity as the late messenger RNA shows only one open reading frame for translation. Considering that VP3 corresponds to the carbosyl terminal part of VP2, and considering various evidence which indicates that the SV40 Hind E segment is part of the amino acid sequence of VP2-VP3. It continues clockwise in Hind K, where it terminates with a UAA signal. The latter is located 110 nucleotides beyond the initiation signal for the major structural protein VP1 (Fiers et al., 1975; Van de Voorde et al., 1976). Hence this small overlapping region of the genome codes for the synthesis of three different proteins in two different reading frames. The deduced amino acid sequence covers a major part of the vp3 poly peptide, and the amino acid composition is in good agreement with published values (Greenaway and Levine, 1973).

Nucleotide sequence of simian virus 40 Hind H restriction fragment.

The restriction fragment Hin d H contains 5·2% of the genome of simian virus 40 (SV40) and is located near the middle of the early region. It can be split by the Arthrobacter luteus (Alu) enzyme into fragments Hin d H-A 1 and Hin d H-A 2 . The nucleotide sequence of fragment Hin d H is reported here. It has been established by analysis of transcription products, synthesized by Escherichia coli RNA polymerase and nucleoside triphosphates, one of which was (α- 32 P)-labeled. These products are very heterogeneous in size and may even exceed the length of the template. Strand assignment was possible by hybridizing the asymmetric, labeled transcript of total SV40 DNA to filter-bound Hin d H DNA. Very clean, discrete products were obtained under appropriate conditions where transcription was dependent on an added primer such as (Ap) 5 A. These transcripts were derived from one strand only, except in the case of Hin d H-A 2 , where the product (in a single chain) contained information derived from both strands. Unambiguous confirmation of the sequence was obtained by experiments directly on the terminally labeled DNA fragments. The message strand is particularly Ap-rich (37%) and purine rich. The dinucleotide CpG occurs only once but UpC is also rare. The nucleotide sequence can be translated unambiguously into an amino acid sequence. This polypeptide, which is part of the gene A -protein, is neutral, rich in methionine, cysteine and tyrosine, and has a high lysine to arginine ratio. All serine codons are of the A-G-Py type; A and U are clearly preferred as third bases.

Location of the small restriction fragments, Hind-L, Hind-M and Hpa-E, on the simian virus 40 genome

Abstract Digestion of simian virus 40 (SV40) DNA (strain 776) with the restriction enzymes from Hemophilus influenzae Rd (Hind II + III) produces, in addition to 11 major fragments two small pieces, Hind-L and Hind-M. These correspond to approximately 30 and 20 base-pairs, respectively. The order on the genome was found to be Hind-C-Hind-L-Hind-M-Hind-D. Digestion of SV40-DNA with the H. parainfluenzae enzymes (Hpa I + II) procedures in addition to the four major fragments a small piece Hpa-E. The latter is identical to Hind-M.

A new restriction endonuclease from Acelobacter pasteurianus

A restriction endonuclease, ApaI, has been partially purified from Acetobacter pasteurianus. This enzyme cleaves bacteriophage lambda DNA and Simian virus 40 DNA at one site, adenovirus-2 DNA at more than nine sites, but it does not cleave phi X174 DNA nor plasmid pBR322 DNA. This enzyme recognizes the sequence (formula; see text) and cuts at the sites indicated by the arrows.

A new restriction endonuclease fromAcelobacter pasteurianus

A restriction endonuclease, ApaI, has been partially purified from Acetobacter pasteurianus. This enzyme cleaves bacteriophage lambda DNA and Simian virus 40 DNA at one site, adenovirus-2 DNA at more than nine sites, but it does not cleave phi X174 DNA nor plasmid pBR322 DNA. This enzyme recognizes the sequence (formula; see text) and cuts at the sites indicated by the arrows.