A T3 and T7 Recombinant Phage Acquires Efficient Adsorption and a Broader Host Range

It is usually thought that bacteriophage T7 is female specific, while phage T3 can propagate on male and female Escherichia coli. We found that the growth patterns of phages T7M and T3 do not match the above characteristics, instead showing strain dependent male exclusion. Furthermore, a T3/7 hybrid phage exhibits a broader host range relative to that of T3, T7, as well as T7M, and is able to overcome the male exclusion. The T7M sequence closely resembles that of T3. T3/7 is essentially T3 based, but a DNA fragment containing part of the tail fiber gene 17 is replaced by the T7 sequence. T3 displays inferior adsorption to strains tested herein compared to T7. The T3 and T7 recombinant phage carries altered tail fibers and acquires better adsorption efficiency than T3. How phages T3 and T7 recombine was previously unclear. This study is the first to show that recombination can occur accurately within only 8 base-pair homology, where four-way junction structures are identified. Genomic recombination models based on endonuclease I cleavages at equivalent and nonequivalent sites followed by strand annealing are proposed. Retention of pseudo-palindromes can increase recombination frequency for reviving under stress

Characterisation data of simple sequence repeats of phages closely related to T7M

Coliphages T7M and T3, Yersinia phage ϕYeO3-12, and Salmonella phage ϕSG-JL2 share high homology in genomic sequences. Simple sequence repeats (SSRs) are found in their genomes and variations of SSRs among these phages are observed. Analyses on regions of sequences in T7M and T3 genomes that are likely derived from phage recombination, as well as the counterparts in ϕYeO3-12 and ϕSG-JL2, have been discussed by Lin in “Simple sequence repeat variations expedite phage divergence: mechanisms of indels and gene mutations” [1]. These regions are referred to as recombinant regions. The focus here is on SSRs in the whole genome and regions of sequences outside the recombinant regions, referred to as non-recombinant regions. This article provides SSR counts, relative abundance, relative density, and GC contents in the complete genome and non-recombinant regions of these phages. SSR period sizes and motifs in the non-recombinant regions of phage genomes are plotted. Genomic sequence changes between T7M and T3 due to insertions, deletions, and substitutions are also illustrated. SSRs and nearby sequences of T7M in the non-recombinant regions are compared to the sequences of ϕYeO3-12 and ϕSG-JL2 in the corresponding positions. The sequence variations of SSRs due to vertical evolution are classified into four categories and tabulated: (1) insertion/deletion of SSR units, (2) expansion/contraction of SSRs without alteration of genome length, (3) changes of repeat motifs, and (4) generation/loss of repeats. Keywords: Simple sequence repeats, T7M, Bacteriophage genome, SSR variability classificatio

DETAILED CHARACTERIZATION OF OVOMUCOID THIRD DOMAINS AND OF THEIR INTERACTION WITH ENZYMES

Our laboratory is working on a sequence to reactivity algorithm for protein inhibitors of serine proteinases in general and for avian ovomucoid third domains in particular. The algorithm is a set of rules for determining the enzyme-inhibitor association equilibrium constants, K(,a)(\u27obs), from the amino acid sequence. To continue this research we need many purified third domain variants of specified amino acid sequences. Therefore, sensitive techniques for separating variants and methods for generating specified new variants are desired. This thesis addresses both of these problems. A highly sensitive HPLC technique was developed to separate pairs of ovomucoid third domains with closely related sequences. Even when two third domains differ at a single uncharged residue, such as Ser/Gly, they were separated to the base line by reverse phase HPLC on (mu)Bondapak C(,18) columns with a binary gradient including H(,2)O, trifluoroacetic acid and organic solvent. The detector was operated at 214nm. Separations of protein mixtures in this system can be achieved with loads of 1.6(mu)g to 1mg. Proteins differing in length of the chain by one or two (out of 56) neutral amino acids were also resolved. The semisynthetic method has been used to obtain ovomucoid third domain variants with chymotrypsin inhibitory specificity (Wieczorek and Laskowski, 1983). It was applied here to semisynthesize variants with trypsin inhibitor activity. In order to test specific parts of the algorithm, two hybrid third domains that have Lys at reactive site and differ only by a single Gly/Asp change at P(,14)\u27 position were generated. The final yield of the semisynthetic protein was approximately 11%. The P(,14\u27) Gly/Asp change has no effect on Ka(\u27obs)\u27s of the hybrids with (beta)-trypsin or Streptomyces griseus proteinase B (SGPB). However, the inhibitor with P(,14\u27) Gly is 7 fold stronger for Streptomyces griseus proteinase A (SGPA). The association constants of the hybrids show a P(,1), P(,14\u27) minor non-additivity for SGPA and a P(,1), P(,14\u27), major nonadditivity for SGPB. Hypotheses explaining these two non-additivities in detailed molecular terms are proposed

Managements of giant cell tumor within the distal radius: A retrospective study of 58 cases from a single center

Background: Giant cell tumor of bone (GCTB) in distal radius is a benign but invasive bone tumor characterized by strong aggressive behavior and frequent recurrence. Methods: To identify recurrence related risk factors and decide suitable surgical strategy, the potential tumor- and treatment-specific factors, post-operative oncologic and functional outcomes were collected and analyzed from 58 patients with GCTB of the distal radius at our center. Results: With the numbers available, our analysis strongly indicated soft tissue extension (with vs. without, HR: 5.645, 95% CI: 1.424 to 22.377, p = 0.014) and size of GCTB (diameter ≥ 5 cm vs. 5 cm HR: 3.893, 95% CI: 1.109 to 13.659, p = 0.034) are the two independent risk factors related to local relapse. Neither surgical procedures (curettage vs. en-bloc resection) nor other factors apparently affected the recurrence, including age, tumor nature, dominant hand involvement, pathological fracture conditions or pre-operative denosumab. However, intralesional curettage group achieved much better functional scores ((VAS: 2.5 ± 0.8 vs. 3.6 ± 1.2, p = 0.011; MSTS: 20.2 ± 3.4 vs. 16.7 ± 3.8, P = 0.034; DASH 9.1 ± 3.9 vs. 16.4 ± 5.5, p = 0.030) and much less complications (non-unions, dislocations, fractures and infections) compared to resection ones. Furthermore, denosumab provided dramatic pain reduction and strong tumor suppression, facilitating curettage with local adjuvants even in GCTB with advanced status. Conclusions: Taken together, the radiographic presentations (soft-tissue extension and tumor size) are the strong prognostic predictors of local recurrence of GCTB in distal radius. In most tumors, an initial treatment with curettage remains feasible and first-choice, especially with the adjuvant denosumab. Keywords: Giant cell tumor of distal radius, Recurrence, Surgical treatments, Denosuma

Efficiency of plating of phages on <i>E. coli</i> female and male strains determined at 37°C.

<p>Efficiency of plating of phages on <i>E. coli</i> female and male strains determined at 37°C.</p

Structures of four-way junctions in gene <i>17</i> of T3 and T7.

<p>(A) T3 nt 33324–33353 (B) T3 nt 33317–33347 (C) T7 nt 35109–35139 (D) T7 nt 35082–35117. One strand is highlighted in grey; the other is not. Arrows indicate Endo I cutting sites.</p

The mechanism of endonucleolytic cleavages at nonequivalent sites and strand annealing (CNSA).

<p>Colors and arrows are the same as those of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030954#pone-0030954-g005" target="_blank">Figure 5</a>. The steps are: 1, production of DSBs by cutting at nonequivalent sites of T3 and T7 DNA; 2, 5′ resections of DSBs of T3 and T7; 3, annealing of the T3 DSBs with the T7 DSB; 4, removal of the nonhomologous nucleotides, filling the gap, and ligation.</p