Gene VI Research Articles

Filamentous phage DNA cloning vectors: A noninfective mutant nonh a nonpolar deletion in gene III

A filamentous phage derivative, fKN16, has been constructed from the tetracyclineresistance transducing phage fd-tet by deleting a 507-base-pair (bp) segment of phage gene III. In accord with the importance of the gene III protein in infection, the infectivity of fKN16 phage is less than 10 −8 that of fd-tet phage. In contrast to most gene III amber mutants, which are polar on the downstream phage genes VI and I, fKN16 should be a nonpolar mutant since its 507-bp deletion spans an integral number of coding triplets. And indeed, two phage traits that may depend on gene VI and I function—the level of phage production and packaging into unit-length virions—appear to be normal in fKN16. High phage production coupled with very low infectivity make fKN16 suitable as a vector for DNA cloning experiments requiring a high level of biological containment. The characteristics of fKN16 as a vector were investigated in detail, using HindIII fragments of phage λ DNA as model foreign DNA. fKN16 may also be useful in studying the role of the gene III protein in the filamentous phage life cycle.

Isolation and characterization of the C and D proteins coded by gene IX and gene VI in the filamentous bacteriophage fl and fd.

The C and D proteins from bacteriophage fd and fl have been purified and characterized. Since the DNA sequence is known, the amino acid composition of these purified proteins indicated that they are coded for by the phage, the C protein being the product of Gene IX and the D protein specified by Gene VI. The molecular weights of the C and D proteins were calculated from the DNA sequences to be 3,650 and 12,350, respectively. These values are close to the molecular weights observed after polyacrylamide gel electrophoresis of the proteins in sodium dodecyl sulfate. Since the C and D proteins can be selectively labeled with radioactive cysteine and arginine, it was possible to estimate the number of the C and D protein molecules relative to the number of A protein molecules which had been accurately determined in previous studies. Based on these results, the average phage particle contains 5 A, 5 D, and 10 C protein molecules.

Nucleotide sequence of the genes III. VI and I of bacteriophage M13

A DNA region of 2750 base pairs encompassing the genes III, VI and I of bacteriophage M13 has been sequenced by the Maxam-Gilbert procedure. By establishing the nucleotide changes introduced by several amber mutations, the coding region and the regulatory signals of each gene have been deduced. The genes appear to span 1275 base pairs (gene III; mol.wt. 44,748) 339 base pairs (gene VI; mol.wt. 12,264) and 1047 base pairs (gene I; mol.wt. 39,500). Their separating non-codogenic regions are extremely short, namely two and one base pair, respectively. The C-terminal end of gene I, however, intrudes 23 nucleotides into gene IV. From the nucleotide sequence it appears that the minor capsid protein of the phage, which is encoded by gene III, is synthesized in a precursor form containing 18 extra amino acids at its N-terminal end. Furthermore, in this capsid protein two clusters of a fourfold repeat of the sequence Glu-Gly-Gly-Gly-Ser are apparent. Gene VI appears to code for a small, extremely hydrophobic polypeptide. Its total hydrophobic amino acids content of 51% suggests that this protein can only function in the host cell membrane.

Transcription of bacteriophage M13 DNA: existence of promoters directly preceding genes III, VI, and I.

In vitro transcription and coupled transcription-translation studies have been performed with restriction fragments of bacteriophage M13 replicative-form DNA which contain either gene III, gene VI, or gene I. It could be demonstrated that DNA fragments which contain gene III were able to direct the synthesis of gene III protein. Fragments which encompassed genes VI and I gave rise to the synthesis of gene I protein only, whereas gene I-containing fragments were able to direct the synthesis of gene I protein. None of the fragments studied gave rise to a detectable level of gene VI protein, although an RNA transcript of gene VI could readily be obtained during in vitro transcription of the relevant gene VI-containing DNA fragments. From these results we have concluded that the promoters A0.44 and A0.49 are located in front of genes VI and I, respectively, and that gene III is also equipped with a promoter (X0.25). Introduction of a single cleavage within the gene III region does not abolish the expression of genes VI and I in vitro. Hence, the expression of these genes is not solely dependent on the initiation of RNA synthesis at the gene III promoter or on leakage of transcription through the central termination site (T0.25), but is also determined by the initiation frequency of RNA synthesis at their individual promoters.

Mapping of bacteriophage f1 ribosome binding sites to their cognate genes

We have isolated ribosome binding sites from bacteriophage f1-mRNA transcripts. These ribosome binding sites were hybridized to purified “minus”-strand restriction fragments, isolated and subjected to two-dimensional fingerprint analysis following digestion with RNase T 1. By this method, we have mapped the three ribosome binding sites sequenced by Pieczenik et al. [Pieczenik, G., Model, P., and Robertson, H. D. (1974). J. Mol. Biol. 90, 191–2141 to fl genes VIII, V, and IV, respectively, and have identified an additional ribosome binding site which maps to either genes VI or I.

Mapping of promoter sites on the genome of bacteriophage M13.

With the aid of transcription studies on restriction fragments of bacteriophage M13 DNA it has been demonstrated that at least eight promoter sites are located on the M13 genome. Five of these promoters initiate the synthesis of RNA chains which contain at their 5'-terminal end pppG (G promoters), while the other three promoters initiate RNA chains which start exclusively with pppA (A promoters). The positions of these promoter sites on the physical map are: 0.82 (G0.82), 0.88 (G0.88), 0.94 (G0.94), 0.01 (G0.01), 0.08 (G0.08), 0.36 (A0.36), 0.51 (A0.51) and 0.56 (A0.56). The G promoters were found to be clustered within a distance of one-third of the genome length from the central termination site for transcription (map position 0.77). The A promoters, however, were found at greater distances from this termination signal. Based upon the incorporation of [gamma-32P]ATP or [gamma-32P]GTP, the capacity of these promoters to initiate the synthesis of RNA chains varies. The strongest G promoters are G0.82, G0.94 and G0.08 and the strongest A promoter is A0.36. As judged from their position on the genetic map, it is postulated that two promoters, namely G0.94 and G0.01, are located within gene II. The other promoters are most probably located immediately in front of the gene VIII/VII boundary (G0.82), and immediately in front of gene V (G0.88), gene II (G0.08), gene IV (A0.36), gene I (A0.51) and gene VI (A0.56). No evidence has been obtained so far for the existence of a promoter immediately in front of gene III.

Identification and characterization of the in vitro synthesized gene products of bacteriophage M13.

Bacteriophage M13 replicative form (RF) DNA was used to direct coupled transcription and translation in cell-free extracts prepared from Escherichia coli. By using RF DNA, isolated from cells infected with appropriate amber mutants of this phage, it has been possible to identify the products of genes I through IV. By using the same methods no gene-product relationship could be demonstrated for genes VI and VII. Coupled in vitro protein synthesis studies on RF-III DNA, a linear double-stranded DNA molecule, obtained after cleavage of either RF-I or RF-II DNA with the restriction endonuclease R.Hin11 from Haemophilus influenzae, indicated that the cleavage site for this enzyme is located in gene II. The in vitro products of both gene III and gene VIII are about 30 and six amino acids longer, respectively, than their native counterparts present within the virion. These results suggest that the latter proteins arise in vivo by cleavage of precursor molecules. Coupled transcription and translation studies on a DNA fragment which only contained the genetic information coding for gene IV protein, obtained after cleavage of RF DNA with the restriction endonuclease R.Hap11 from Haemophilus aphirophilus, indicated that a large number of the in vitro synthesized polypeptides are the result of premature chain termination.

In vitro synthesis of bacteriophage f1 proteins

Covalently closed, circular, double-stranded DNA isolated from cells infected with bacteriophage f1 has been used as a template for coupled transcription and translation in vitro. By using appropriate amber mutants of the phage, it has been possible to identify six gene-specific polypeptides. Three of these correspond to previously known proteins (products of genes III, V and VIII), while three others had not been previously identified (products of genes I, II and IV). No polypeptides could be identified as the products of genes VI and VII. The relative amounts of the various polypeptide products are highly sensitive to the concentration of magnesium ions, somewhat sensitive to the concentration of monovalent cations, but are not affected by the introduction of a limited number of single-strand breaks into the template. No differences could be detected between the profile of products made in extracts of phage f 1 -infected cells when compared with the profile of products made in extracts of uninfected cells. Suppression of the amber mutations by Su + tRNA is demonstrated. Additionally, however, Su + tRNA added to the in vitro system promotes the synthesis of an additional polypeptide not found in the presence of Su − tRNA. This phenomenon is independent of the genetic constitution of the template, and is postulated to result from the suppression of a weak terminator.

DNA synthesis in nucleotide-permeable Escherichia coli cells : II. Synthesis of replicative form DNA of phage φX174

Escherichia coli cells were treated with mitomycin C to suppress host DNA synthesis, infected for five minutes with bacteriophage φX174, made permeable to nucleotides by treatment with ether and allowed to synthesize DNA from exogenous deoxynucleoside triphosphates. The product was 70 to 80% replicative form (RF) DNA of the phage according to sedimentation and annealing properties. RF synthesis occurred at the normal level in DNA polymerase-deficient cells and did not occur in these cells when they were infected with a gene VI amber mutant of the virus, which in the living cells does not replicate beyond the first RF. The structure of intermediates of RF synthesis in ether-treated cells is thought to be basically consistent with the rolling circle model of RF replication proposed by Knippers, Whalley & Sinsheimer (1969). In addition, the results suggest that a temporary interruption exists in the complementary strand just ahead of the growth point. Complementary strand material, and possibly also viral strand material, exists early after synthesis as short pieces having only a small fraction of the viral length. These pieces are subsequently joined together but can be induced to accumulate. This accumulation is suppressed by 0.2 m m-dATP but not detectably by any of the other deoxynucleoside triphosphates in high concentration, or by ATP or DNA ligase cofactor. Label in RF can be chased up to 80% into supertwisted molecules.