Figure 1.
Genome organization of tf and its comparison with LUZ24 bacteriophage.
Predicted genes are designated with arrows indicating their directions of transcription. Color and symbol codes adopted are shown at the bottom of the figure. The similarities between corresponding amino acid sequences of tf and LUZ24 are indicated by connecting lines.
Figure 2.
(A) Automated sequencing of the bacteriophage tf DNA at termini regions. Alternative end-nucleotides are indicated. (B) Results of the primer extension analysis of the 5′-ends in the tf DNA. Nucleotide positions corresponding to the stoppages at the phage DNA ends are indicated with arrows; black arrow for the position corresponding to the major band and gray arrows – position corresponding to minor bands at the left terminus only. (C) Analysis of the termini junction in the circularized tf DNA. Bacteriophage DNA was treated with T4 DNA polymerase and circularized using T4 DNA ligase. The junction region was analyzed by automated sequencing of the corresponding PCR product as described in the text. The junction identified is shown on the chromatogram by a vertical line and the schematic of the ligated genome ends is shown above the chromatogram. (D) Analysis of the 3′-ends in the tf DNA. Denatured tf DNA was treated with TdT enzyme in separate experiments in the presence of either dATP or dTTP and PCR products were obtained using resulting preparations and one phage specific and one poly-dT (poly-dA) specific primer. Chromatograms for all types of experiments are presented with the corresponding sequences shown at the bottom of the figure. Terminal sequences of the phage genome are shown by shading. (E) Structure of the tf DNA ends’ sequences.
Figure 3.
Identification and analysis of the localized nicks in tf DNA molecules.
DNA preparation of bacteriophage tf was denatured in 0.1 M NaOH and subjected to electrophoresis in 0.9% agarose gel; in a control experiment a DNA from the same preparation was treated with T4 DNA ligase prior to denaturing in 0.1 M NaOH and electrophoresis.
Table 1.
Putative nickase recognition sites in the genomes of LUZ24-like phages.
Figure 4.
Primer extension analysis of the sci-14.
The schematic of the experiment is shown at the top of the Figure: non-ligated (1) and ligated (2) DNA preparations were digested with HaeIII restriction endonuclease and primer extension reaction were performed using the same [32P] labeled primer. The results of primer extension with Klenow fragment (PE:K) and Taq DNA polymerases (PE:T) are presented at the bottom part of the Figure. Bands corresponding to the stoppages at the sci-14 and HaeIII sites are indicated by arrows. Sequencing ladders (A, G, C, T) were generated by Sanger sequencing of the T4 DNA ligase treated tf DNA employing the same [32P] labeled primer used in primer extension.
Figure 5.
Comparison of localized nick (sci) distribution in genomes of the phages T5, phikF77 and tf.
Nicks are shown as long vertical lines. Only “Major” nicks are shown in T5 chromosome. Direct terminal repeats (DTR) found in these phages are indicated with arrows. Locations of all nicks are given as the percentages of the corresponding genome size.
Figure 6.
Electron Microscopy of partially denatured tf DNA molecules.
Nicks are visualized as partially denatured regions which are indicated by black arrows. Bacteriophage head attached to DNA molecule is indicated by white arrow.
Figure 7.
Putative processing of the tf DNA from concatemer substrate.
Regions on the concatemer containing terminase recognition sequences are boxed with a red box corresponding designating sequences found at the left end of the genome and a green one – at the right. A putative core recognition site for the terminase enzyme is shaded with grey and positions of cuts are indicated by vertical arrows. Palindrome sequences identified within these sites are shown by horizontal arrowed lines.