T4 DNA Molecules Research Articles

Terminal repetition in non-permuted T3 and T7 bacteriophage DNA molecules

Evidence in the preceding paper indicates that the DNA molecules from bacteriophage T2 contain terminally repetitious nucleotide sequences. In the first part of this paper, we describe similar, but more extensive, experiments performed on T3 and T7 DNA molecules. Partial degradation of either phage DNA molecule by exonuclease III allows efficient conversion of the linear molecules into circular ones. Fragments of these molecules, which are not expected to be terminally repetitious, can be partially degraded under identical conditions, but they do not anneal to form circles. These facts indicate that T3 and T7, like T2 DNA, have terminally repetitious sequences. The second part of the paper deals with the non-permuted character of T3 and T7 DNA molecules. The complementary strands of each species of molecule were completely separated with alkali and then annealed. In both cases, this treatment produced predominantly linear duplex molecules of a length equal to that of the undenatured molecules. From this we conclude that the collection of molecules is non-permuted (i.e., they are a unique collection of sequences). When this experiment is taken together with earlier equivalent experiments on T2 DNA molecules, which are readily cyclized by this treatment, it appears that alkali denaturation and annealing is a rigorous test for the presence of multiple permutations. In sum, T3 and T7 DNA molecules are terminally repetitious but non-permuted; T2 DNA is terminally repetitious and permuted.

The anatomy of the T5 bacteriophage DNA molecule

The T5 bacteriophage DNA molecule, unlike the molecules from T2, P1, λ and T7 bacteriophage, contains interruptions at specific locations in both of its polynucleotide chains. When the molecule is denatured, it gives rise to polynucleotide chains which sediment in at least four different zones. By contrast, denatured T2, P1, λ and T7 DNA sediment as unimodal zones. The lengths of the polynucleotide chains -which comprise these zones were calculated through use of a relationship established for determining the relative lengths of polynucleotide chains from their rate of sedimentation in alkaline sucrose gradients. The positions of the interruptions along the molecule are under the genetic control of the phage and apparently are not a property of the host. The phenomenon is seen in another phage, PB, which is closely related to T5. The interruptions in the molecule are probably gaps in the polynucleotide chains. This conclusion was reached because the chains are not intact when the molecule is denatured in formamide, a mild organic denaturing agent. A number of models were proposed for ways in which the six polynucleotide chains could be arranged to form the intact duplex molecule. Two reannealing experiments were performed which confirmed some general features which all of the models had in common. The existence of gaps in the T5 DNA molecule helps to explain some earlier observations on the structure of the molecule and suggests interpretations for other results concerning its biological function.

Capacity of T4 DNA to serve as template for purified Escherichia coli RNA polymerase

The dependence of in vitro RNA synthesis, catalyzed by purified Escherichia coli RNA polymerase, on the concentration and secondary structure of T4 DNA templates in the reaction mixture has been studied by means of kinetic and sedimentation experiments. The following results were obtained. (1) If the weight ratio of DNA to polymerase in the reaction mixture is low, then the rate of RNA synthesis is proportional to the DNA concentration and nearly independent of the polymerase concentration (saturation of DNA with polymerase). If that ratio is high, then the rate of RNA synthesis is proportional to the polymerase concentration and nearly independent of the DNA concentration (saturation of polymerase with DNA). For intermediate ratios, the ensuing rate of RNA synthesis in the reaction mixture can be increased by addition of either more polymerase or more DNA, suggesting that both polymerase and starting sites for polymerase on the DNA are heterogeneous with respect to their mutual binding affinities. (2) The rate of chain growth of the RNA is independent of the DNA concentration; accordingly, at limiting DNA concentration and fixed polymerase concentration, only the number of polymerase molecules becoming active is increased with increasing DNA concentration. (3) The maximum number of long RNA molecules that can be synthesized simultaneously, and thus the maximum number of polymerase molecules that can be fully active per native T4 DNA unit, is estimated to be 180. (4) On heat-denatured T4 DNA, the maximum number of RNA molecules that can be synthesized per unit weight of DNA is several times higher than on native T4 DNA. To account for these results, a model is proposed which assumes a limited number of specific starting sites for RNA polymerase on the native T4 DNA molecule.

Preferred breakage points in T5 DNA molecules subjected to shear

T5 DNA molecules subjected to critical rates of shear break into two pieces measuring 0.6 and 0.4 of the original molecular length. The fragments of length 0.6, isolated and subjected to a higher rate of shear, break into two pieces measuring 0.4 and 0.2 of the original molecular length. The molecules must contain two to four sites of preferred breakage at prescribed locations. Preferred breakage points have not been detected and may be absent in DNA's of some other species.

MAPPING OF TEMPERATURE-SENSITIVE MUTANTS IN BACTERIOPHAGE T5.

5, one of the classical T phages of Escherichia coli, has been the subject of many studies, but its genetic analysis has been sporadic. Beginning with heatstable ( s t ) mutants (ADAMS and LARK 1950; ADAMS 1953), there have been reports of mutants with altered host range (LANNI and LANNI 1956), serological behavior (LANNI and LANNI 1957), lysis-inhibiting properties (U. T. LANNI 1958), and buoyant density and sedimentation rate (HERTEL, MARCHI and MULLER 1962; LARK 1962). Host-range and serological differences are correlated, as are differences in heat-stability and density. Several of the aforementioned mutants show altered plaque type. Additional plaque-type mutants of various kinds are easily isolated from routine assay plates, especially with E. coli strain F as the host. Genetic recombination in T5 was first reported by ADAMS (1951, 1952) in crosses between T5 and the taxonomically related phages PB. BG3, and 29 alpha. which were independently isolated from natural sources. T5, PB, and their hybrids have been used in studies of the cytological effects of phage infection (MURRAY and WHITFIELD 1953) and the genetic control of host-range and serological specificity ( FODOR and ADAMS 1955; FODOR 195 7; WASSERMANN 1959; see also F. LANNI 1958). The considerable mutual exclusion in mixed infection and the multiple hereditary differences between the parents limit the value of these interstrain crosses for genetic mapping. More recently, HERTEL et al. (1962) reported recombination between plaque-type and density markers in T5 mutants, and NI. NESSON (personal communication from R. S. EDGAR) observed recombination between temperature-sensitive ( t s ) mutants of the kind to be described here. (Note: The st phenotype involves an increased stability of the free phage particle in media of low ionic strength, whereas the ts phenotype involves a decreased ability to grow at a moderately elevated temperature). A suitably comprehensive genetic map is needed in conjunction with studies of T5 progressing in various laboratories and in comparative bacterial virology. For example, under certain well defined conditions the T5 DNA molecule can be sectioned artificially during transfer from the infecting phage particle to the host cell. The transferred section amounts to about 8 percent of the whole DNA

Molecular aspects of DNA transfer from phage T5 to Host Cells: II. Origin of first-step-transfer DNA fragments

We have previously proposed that blending of early bacteriophage T5— E. coli complexes ( FST complexes ) results in the scission of phage DNA molecules partially transferred to host cells. Such blended complexes should then retain only fragments of the ingoing phage DNA. Blending of late phage—bacterium complexes ( stabilized complexes ), in which DNA transfer has been completed, should not break the phage DNA molecules. With a method of extraction which rninimizes DNA breakage, followed by zone centrifugation of the crude extracts, we find that extracts of blended FST complexes contain almost exclusively T5 DNA fragments ( FST fragments ) with a size 8·3±1% that of intact T5 DNA molecules, whereas extracts of unblended FST complexes, as well as extracts of blended or unblended stabilized complexes, contain mostly intact T5 DNA molecules. The results rule out the possibility that the FST fragments arise by multiple breakage of a few totally transferred DNA molecules. Instead, they indicate, in support of our hypothesis, that each adsorbed phage particle of an FST complex yields a single FST fragment on blending.

The Arrangements of Nucleotide Sequences in T2 and T5 Bacteriophage DNA Molecules

The genetic map of T4 (and T2) bacteriophage is circular but the DNA molecule that is liberated by phenol extraction is a linear duplex of polynucleotide chains. If the genetic map is related to the physical structure of the DNA molecule, the problem arises as to how a linear molecule can give rise to a circular map. An explanation can be made on the basis that the bacteriophage liberate molecules which have nucleotide sequences which are circular permutations of each other. Thus, markers which are most distant on one molecules are closest together on another. To test this hypothesis, the middles of T2 and T5 DNA molecules were mechanically deleted and the absence of certain nucleotide sequences was tested by "renaturation" or "reannealing" experiments using columns containing denatured DNA immobilized in agar beads. The results indicate that when the middles are deleted from the T5 DNA molecule, some special sequences are removed; whereas, when the middles are deleted from the T2 DNA molecule, no special group of sequences is removed. This would indicate that T2 molecules begin at different points in their nucleotide sequence, while T5 molecules all begin at the same point. It is likely that this permutation of sequences of T2(T4) molecules is related to the circularity of their genetic map.

The Arrangements of Nucleotide Sequences in T2 and T5 DNA Molecules

The DNA molecule which is liberated from T2 or T4 bacteriophage is a linear duplex over most or all of its length. The genetic map of T4 (and presumably T2) is circular as shown by Streisinger, Edgar, and their collaborators, cited by Luria (1962, Ann. Rev. Microb. 16: 205–240). If genetic structure is a simple reflection of physical structure, then a problem arises: exactly how can a linear DNA molecule produce a circular genetic map? The suggestion has been made that the nucleotide message begins at different points in different molecules. This would mean that the sequences of nucleotides in the linear molecules liberated from an “isogenic” phage stock would be circular permutations of one another. Thus, markers most widely separated in one phage would be most closely linked in another and such a model might be able to account for the circular map. Dr. Irwin Rubenstein and I attempted...

Sedimentation Coefficient and Fragility under Hydrodynamic Shear as Measures of Molecular Weight of the DNA of Phage T5

T5 DNA molecules resemble fragments of T2 DNA of molecular weight 84 x 10(6) with respect to sedimentation coefficient and susceptibility to breakage under hydrodynamic shear. The sedimentation coefficient falls by the same factor when either T2 or T5 DNA is broken at its characteristic critical shear rate. At a given high rate of shear, both DNA's are broken into fragments exhibiting the same sedimentation coefficient. It follows that 84 x 10(6) is a proper estimate of the molecular weight of T5 DNA, and that particles of phage T5, like those of T2, contain a single DNA molecule.