The 5,577 nucleotide long sequence of bacteriophage G4 DNA has been determined using the 'plus and minus' and chain termination methods of DNA sequencing. This sequence has been compared with that of the closely related bacteriophage phiX174 (refs 1, 55). In the coding regions there is an average of 33.1% nucleotide sequence differences between the two genomes, but the distribution of these changes is not random and the sequence of some genes is more conserved than others. There is less sequence similarity between the untranslated intergenic regions of G4 and phiX174, but despite this the sequences of the J/F, F/G and H/A untranslated spaces in both genomes have similar sized hairpin loops, which may be related to their function.

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Amino acid sequence and composition data of Escherichia coli dnaG primase protein and its tryptic peptides have confirmed that the dnaG gene contains an unusually high number of codons that are not frequently used in most E. coli genes. In 25 E. coli proteins analyzed the codons AUA, UCG, CCU, CCC, ACG, CAA, AAT, and AGG are infrequently used, occurring as 4% of the total codons in the reading frame and 11% and 10% in the nonreading frames. In dnaG they occur as 11% in the reading frame and 12% in the nonreading frames. The rpsU and rpoD genes, which flank the dnaG gene [Smiley, B. L., Lupski, J. R., Svec, P. S., McMacken, R. & Godson, G. N. (1982) Proc.Nati Acad Sci USA 79, 4550-4554], however, have normal codon usage. Translational modulation using isoaccepting tRNA availability may therefore be part of the mechanism of keeping the dnaG gene expression low, while expression of the adjacent rpsU and rpoD genes on the same mRNA transcript is high.Nucleotide sequences, which are becoming available in increasing numbers, have clearly indicated that codon usage is not random and that, within an organism, certain synonymous codons are preferred to others. The spectrum ofsynonymic codon preference appears to differ from organism to organism and this has now been shown to reflect the pattern of tRNA isoaccepting species expressed in that organism (1).Because there are frequently and less-frequently expressed members of tRNA isoaccepting species within a cell, the lessfrequently expressed tRNAs can theoretically be used to modulate translation of protein expression. This was proposed by Fiers and Grosjean from an examination of the codon usage of the bacteriophage MS2 RNA (2) but has never been experimentally verified.It has also been observed that codon usage within the known coding reading frame ofa nucleotide sequence is different from that observed in the two noncoding reading frames. Analysis of codon usage had therefore been proposed by several groups to be a method of identifying the correct coding reading frame within a nucleotide sequence and also as a method ofidentifying nucleotide sequencing errors (3, 4). This paper, however, demonstrates that the Escherichia coli dnaG primase gene does not conform to the codon usage of either the organism or its flanking genes in the macromolecular synthesis operon [dnaG and rpoD (5) and rpsU4K]. The dnaG gene contains an unusually large number of infrequently used synonymic codons (rare codons) and within the gene there are long stretches of nucleotide sequence that contain more rare codons in the coding reading frame than in the noncoding reading frames. The use ofrare codons in the dnaG gene, therefore, may be part of the mechanism to maintain its expression at low copy number compared with the flanking genes on the same mRNA transcripts as shown by Smiley and colleagues (5,6). This is supported by the observation that the E. coli repressor genes lacI, trpR, and araC, which are also expressed in low amounts, contain unusually high numbers of rare codons.MATER...

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The regulatory regions for the rpsU-dnaG-rpoD macromolecular synthesis operon have been fused to a structural gene whose product is readily assayed (the Cmr structural gene coding for chloramphenicol acetyl transferase, CAT). The promoters (P1, P2, P3, Pa, Pb, Phs) for the macromolecular synthesis operon have different strengths as shown by their relative abilities to drive expression of the CAT gene. Promoter occlusion by P1 can be demonstrated within this operon. Regions 5kb upstream have a profound effect on operon gene expression. There is a thermoinducible promoter located within the dnaG structural gene. One of the macromolecular synthesis operon promoters is under lexA control. Although the operon structure allows coordinate expression of rpsU, dnaG and rpoD these additional features suggest that expression of individual genes can be independently regulated in response to altered growth conditions.

The malarial sporozoite, the infective stage found in the salivary gland of the insect vector, bears highly immunogenic surface antigen(s). Repeated exposure to irradiated sporozoites induces protection against malaria in several host species, including man. Further, monoclonal antibodies that confer passive immunity react with the immunogenic surface determinants of different sporozoite species. One approach to prevent malaria, therefore, would be to produce a vaccine that induces high titres of circulating antibodies against the sporozoite surface determinant(s). However, production of such a vaccine has not been possible since sporozoites cannot be cultivated in vitro and, therefore, only limited amounts of surface antigen may be obtained. To overcome this problem, we have prepared mRNA from Plasmodium knowlesi-infected mosquitoes to construct a cDNA library. From this library we have isolated a clone that expresses the sporozoite surface antigen as a beta-lactamase fusion protein in the plasmid pBR322. This is the first potentially protective malarial antigen to be cloned by recombinant DNA technology.

Complementary DNA clones that code for the immunogenic region of the Plasmodium knowlesi circumsporozoite protein were shown to contain a tandemly repeating 36-base pair unit. A synthetic dodecapeptide corresponding to the predicted reading frame of the repeating nucleotide unit behaved, in an immunoradiometric assay, identically with the native P. knowlesi circumsporozoite protein. The repeating 36-base pair unit occurred 12 times within the gene and accounts for at least one-third of the amino acid sequence of the surface antigen protein.

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