Rudolf Hausmann

Bacteriophage T7 genetics.

It is generally taken for granted that the study of a subject under review has been sanctioned by the scientific community and therefore no further justification is needed. After 30 years of hectic phage genetics, it might, however, be appropriate to pause for a moment in order to evaluate the original motives and aims: why study the genetics of phage, and why, particularly, that of T7?

The gene for Klebsiella bacteriophage K11 RNA polymerase: Sequence and comparison with the homologous genes of phages T7, T3, and SP6

SummaryWe determined the nucleotide sequence of gene 1 of Klebsiella phage K11, which is a member of the T7 group of phages. The largest open reading frame corresponds to a polypeptide with 906 amino acids and a molecular weight of 100383 daltons. The deduced amino acid sequence of this polypeptide shows 71% homology to the T7 RNA polymerase (the product of T7 gene 1), 72% homology to the T3 RNA polymerase and 27% homology to the SP6 RNA polymerase. Divergent evolution was clearly most pronounced in the amino-terminal portion.

In vivo and in vitro “phenotypic mixing” with amber mutants of phages T3 and T7

SummaryGene 17 of phage T7, and a homologous gene of T3, were shown to code for the “serum blocking power” protein (SBP). Noninfective T7 particles lacking gene 17 product (SBP-less particles) could be rendered infective by incubation with extracts of nonpermissive host bacteria infected with amber mutants of T7, as well as of T3, defective in other genes. SBP-less T7 particles activated by extracts of T3-infected cells were characterized as “coat chimeras” by their specificity towards anti-T3 and anti-T7 sera.

One Gene — One Enzyme

Originally, Morgan thought of himself as an embryologist in the quest of “how” — whatever that meant in chemical-physiological terms — an individual evolved out of an egg cell. His students coerced him to become a geneticist. In doing so, they encountered little resistance, especially since the discovery of the sex-linked inheritance of the white-eyed characteristic of the fruit fly Drosophila (Morgan, 1910) had introduced him to the field of genetics. In contrast with embryology, the results obtained in genetics led to good, palpable progress. In the back of his mind, though, he never totally abandoned the wish to grasp the real mechanism of gene action governing the development of an embryo. The hopelessness of that intent — considering the state of the art at that time — could not then be sensed; an understanding of it was only possible in retrospect, from a viewpoint still to be generated in the future. Nonetheless, concrete projects were developed to proceed towards the desired research goal.

T7 infection-dependent selective expression of cloned genes in P1-lysogenic Escherichia coli

Expression systems based on the selective transcription of genes cloned behind a T7 promoter, by T7 RNA polymerase, display a non-negligible basal expression when the T7 RNA polymerase gene is present within the host organism before induction of the system. This is a problem, especially for cloning and controlled expression of genes toxic to the host organism. We have circumvented this problem by taking advantage of abortive T7 infection of E. coli (P1), in the course of which T7 RNA polymerase is synthesized but bacterial growth is not quantitatively impaired. We have tested this system with three reporter genes, the 6-phospho-beta-galactosidase gene of Staphylococcus aureus, the luciferase operon of Vibrio harveyi, and the rabbit beta-globin gene; we have found very low basal levels, while, upon T7 infection, transcription is at least as efficient as in other in vivo T7 RNA polymerase systems in use.

Two Groups of Capsule-specific Coliphages Coding for RNA Polymerases with New Promoter Specificities

Four bacteriophages (A16, CK235, phi 1.2 and K31) which specifically attack different encapsulated strains of Escherichia coli have been shown to be related to bacteriophage T7 (which is unable to grow on encapsulated hosts). The conclusion that phages A16 and CK235 are related to T7 is based on similarities in the pattern of expression of intracellular phage proteins, early appearance, in infected host cells, of a phage DNA-specific RNA polymerase and hybridization (albeit to a low extent) of A16 DNA and of CK235 DNA to T7 DNA. The first two criteria also apply to phages phi 1.2 and K31 but hybridization of their DNAs with T7 DNA could not be detected. The RNA polymerases of CK235 and A16 have similar template specificities and the same applies to the RNA polymerases of phi 1.2 and K31. None of the new RNA polymerases can use T7 DNA as template.

Inhibition of gene expression of T7-related phages by prophage P1

SummaryThe gene expression of nine phages of the T7 group was compared after infection of Escherichia coli B(P1). With the exception of phage 13a which grew normally, all of them infected E. coli B(P1) abortively. Differences were found in the efficiency of host killing which ranged from 100% for phage 13a to 37% for phage A1122. Infection by T7 prevented colony formation by about 70% of the cells but they showed filamentous growth until about 2h after infection. It was shown by SDS-polyacrylamide gel electrophoresis and autoradiography of [35S]methionine-labelled phage-coded proteins that all phages except for 13a showed measurable expression only of the early genes. No correlation was observed between killing capacity and the pattern of gene expression, and the ability to hydrolyse S-adenosyl-methionine (SAM, a cofactor for the P1 restriction endonuclease) by means of a phage-coded SAMase. Mixed infection of E. coli B(P1) with 13a and T7 yielded mixed progeny indistinguishable from that observed after mixed infection of the normal host E. coli B. Genetic crosses with amber mutants of 13a and T7 showed that the 13a marker opo+ (overcomes P one), required for growth on B(P1), is located in the early region, to the left of gene 1 (RNA polymerase gene).

Inside the Institut Pasteur — Part II Interrupted Mating

Zygotic induction acquired an additional significance: it hinted at the possibility of analyzing the kinetics of the conjugational events. If Hershey, in a strike of ingenuity, took advantage of a kitchen blender with its shearing power to detach adsorbed phages from their host cells, why not try achieving separation of donor and receptor cells by a similar procedure?

The Code Craze

Whereas the principle of DNA replication was unambiguously revealed by the mere contemplation of its proposed structure, it was clear that the mechanisms by which DNA was supposed to direct protein synthesis would remain thoroughly mysterious for a while to come. How was it possible for DNA to determine the amino acid sequence of proteins, if between the bases of DNA and the amino acids of proteins there was no steric or chemical affinity whatsoever?

The Coronation: DNA Replication

We have by now elaborated a panorama view over the paths leading from the genetic information stored in DNA to the meaningfully regulated transcription of one of its strands into RNA (rRNA, tRNA and mRNA), and through translation of mRNA into polypeptide chains and the folding of these chains into protein molecules with enzymatic activity. These enzymes, in their turn, are the instruments forging new building blocks for renewed macromolecular synthesis. At this point, we reach the peak event of a cell’s life cycle, its coronation: the self-duplication of the genetic information, the replication of DNA.