The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

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Initial sequencing and analysis of the human genome.

We constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. We also constructed a recombinant, pSV0-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells.

With a stepwise degradation and terminal labeling procedure the 3′-terminal sequence of E. coli 16S ribosomal RNA is shown to be Pyd-A-C-C-U-C-C-U-U-AOH. It is suggested that this region of the RNA is able to interact with mRNA and that the 3′-terminal U-U-AOH is involved in the termination of protein synthesis through base-pairing with terminator codons. The sequence A-C-C-U-C-C could recognize a conserved sequence found in the ribosome binding sites of various coliphage mRNAs; it may thus be involved in the formation of the mRNA·30S subunit complex.

https://www.pnas.org/content/pnas/71/4/1342.full.pdf

The 3′-Terminal Sequence of Escherichia coli 16S Ribosomal RNA: Complementarity to Nonsense Triplets and Ribosome Binding Sites

THE early region of the small DNA tumour virus, simian virus 40 (SV40), is known to code for at least two polypeptides, the t and T antigens (‘small t’ and ‘large T’) Both these polypeptides are expressed in cells transformed by the virus1–4, and the T antigen has been shown to be essential for both the initiation and maintenance of the transformed state5–9 We therefore need to know how this T protein interacts with components of the host cell in order to understand the mechanism of SV40-induced transformation We report here that the T antigen in a line of SV40-transformed mouse cells forms an oligomeric complex with a specific cell coded protein

T Antigen Is Bound to a Host Protein in Sv40-Transformed Cells

A SIMPLE method for estimating the most stable secondary structure of an RNA molecule from its sequence was proposed earlier1. This method can be used for predicting and assessing possible secondary structures for recently determined RNA sequences2–4. New experimental5–8 and theoretical9, 10 results allow us to improve the method, without making it more complicated.

Improved Estimation of Secondary Structure in Ribonucleic Acids

The determination of the total 5,224 base-pair DNA sequence of the virus SV40 has enabled us to locate precisely the known genes on the genome. At least 15.2% of the genome is presumably not translated into polypeptides. Particular points of interest revealed by the complete sequence are the initiation of the early t and T antigens at the same position and the fact that the T antigen is coded by two non-contiguous regions of the genome; the T antigen mRNA is spliced in the coding region. In the late region the gene for the major protein VP1 overlaps those for proteins VP2 and VP3 over 122 nucleotides but is read in a different frame. The almost complete amino acid sequences of the two early proteins as well as those of the late proteins have been deduced from the nucleotide sequence. The mRNAs for the latter three proteins are presumably spliced out of a common primary RNA transcript. The use of degenerate codons is decidedly non-random, but is similar for the early and late regions. Codons of the type NUC, NCG and CGN are absent or very rare.

Complete nucleotide sequence of SV40 DNA

Bacteriophage MS2 RNA is 3,569 nucleotides long. The nucleotide sequence has been established for the third and last gene, which codes for the replicase protein. A secondary structure model has also been proposed. Biological properties, such as ribosome binding and codon interactions can now be discussed on a molecular basis. As the sequences for the other regions of this RNA have been published already, the complete, primary chemical structure of a viral genome has now been established.

Complete nucleotide sequence of bacteriophage MS2 RNA: primary and secondary structure of the replicase gene.

By characterization of fragments, isolated from a nuclease digest of MS2 RNA, the entire nucleotide sequence of the coat gene was established. A “flower”-like model is proposed for the secondary structure. The genetic code makes use of 49 different codons to specify the sequence of the 129 amino-acids long coat polypeptide.

Nucleotide Sequence of the Gene Coding for the Bacteriophage MS2 Coat Protein

The nucleotide sequence of the A-protein gene of bacteriophage MS2 has been determined and a model for its secondary structure is proposed. Also the amino acid sequence of the A-protein has been almost completely elucidated.

M. Ysebaert

Papers

Complete nucleotide sequence of SV40 DNA

Complete nucleotide sequence of bacteriophage MS2 RNA: primary and secondary structure of the replicase gene.

Nucleotide Sequence of the Gene Coding for the Bacteriophage MS2 Coat Protein

A-Protein gene of bacteriophage MS2

Total nucleotide sequence of a nearly full-size DNA copy of satellite tobacco necrosis virus RNA