Directionality and Nonreciprocality of Chi-Stimulated Recombination in Phage λ

Chi's are genetic elements that stimulate generalized recombination in their locale in phage λ. All Chi's, wherever located on λ's chromosome, act asymmetrically in crosses blocked in DNA replication: (1) They stimulate exchange primarily to their left on the conventional λ map, and (2) the stimulated exchange is frequently nonreciprocal, the recombinant carrying the Chi element being produced less often than the complementary product.     

Heat induced capsid disassembly and DNA release of bacteriophage λ

Successive structural changes of bacteriophage λ upon heating were characterized with quantitative experimental methods. In the commonly used Tris-Mg buffer, differential scanning calorimetry measurements first established that the protein capsid of λ phage melts at 87 °C and its genomic DNA melts at 91 °C. Interestingly, prior to the capsid melting, λDNA was found to escape out of the capsid and subject to DNase digestion above ~68 °C, as concluded from light scattering, UV absorption, and electron microscopy studies. Further investigations indicated distinct temperature-dependent behaviors of the three phage proteins. Around 68 °C, disruption of the tail first occurs and leads to the escape of λ DNA; above the capsid melting temperature of 87 °C, the auxiliary protein gpD of the phage head remains soluble in solution and resists centrifugal sedimentation, whereas the major capsid protein gpE is easily precipitated and likely exists as aggregates.     

Development of Quantitative Enzyme Immunoassay and Radioimmunoassay of λ-Free Light Chains of Immunoglobulins

New quantitative techniques of radioimmunoassay (RIA) and enzyme immunoassay (EIA) were developed for determination of free light -chains of immunoglobulins (immunometric sandwich-like variants) in biological fluids using two types of monoclonal specific antibodies (MAB-1 and MAB-2) to different epitopes of the antigen molecule: MAB-1 were immobilized on a solid phase (polystyrene beads), whereas MAB-2 were labeled with iodine or with the enzyme. The test systems prepared can be used for determination of concentrations from 25 to 1000 ng/ml, are very sensitive (25 ng/ml), and the analysis time is 5 h. The two methods were compared, and their clinical and diagnostic validity was evaluated in patients with various diseases associated with disorders in the antibody synthesis by the immune system.     

Ubiquitylation of DNA polymerase λ

DNA polymerase (pol) λ, one of the 15 cellular pols, belongs to the X family. It is a small 575 amino-acid protein containing a polymerase, a dRP-lyase, a proline/serine rich and a BRCT domain. Pol λ shows various enzymatic activities including DNA polymerization, terminal transferase and dRP-lyase. It has been implicated to play a role in several DNA repair pathways, particularly base excision repair (BER), non-homologous end-joining (NHEJ) and translesion DNA synthesis (TLS). Similarly to other DNA repair enzymes, pol λ undergoes posttranslational modifications during the cell cycle that regulate its stability and possibly its subcellular localization. Here we describe our knowledge about ubiquitylation of pol λ and the impact of this modification on its regulation.     

Synthesis of amidic alginate derivatives and their application in microencapsulation of λ-cyhalothrin

1-Octyl amine was covalently coupled to sodium alginate(NaAlg) in an aqueous-phase reaction via acidamide functions using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl) as a coupling reagent to provide octyl-grafted amphiphilic alginate-amide derivative(OAAD) for subsequent use in λ-cyhalothrin (LCH) microcapsule application. The structure of OAAD was confirmed by FT-IR and (1)H NMR spectroscopies. The new alginate-amide derivative was used for fabricating microcapsule that can effectively encapsulate LCH by emulsification-gelation technique. The microcapsules were characterized by optical microscopy (OM), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and laser particle size analysis. The encapsulation efficiency and drug release behavior of LCH from the microcapsules were investigated. Results showed that the microcapsules were in spherical form with diameter mostly in the range of 0.5-10 μm and possessed a structure with LCH as core and OAAD as shell. The encapsulation efficiency and the release performance of the microcapsules were influenced by DS of OAAD and amount of CaCl(2). The mechanism of LCH release was found to vary from anomalous to Fickian to quasi-Fickian transport with the DS of OAAD varied from 10.8 to 30.3 and the CaCl(2)/emulsion ratios varied from 0.09 to 0.03%.     

Identity of a Chi site of Escherichia coli and Chi recombinational hotspots of bacteriophage λ

Chi sites in bacteriophage λ stimulate recombination promoted by the RecBC pathway of Escherichia coli. We have located a Chi site within the E. coli lacZ gene by deletion mapping and have isolated a mutation inactivating this Chi. Sequence analysis showed that the mutation arose by a single base-pair transition $\text{G}\text{C}\text{?}\text{→}\text{A}\text{T}\text{?}$ within an eight base-pair sequence (5′ G-C-T-G-G-T-G-G 3′) identical to that found at Chi sites in λ and in plasmid pBR322.     

Mapability of Very Close Markers of Bacteriophage λ

Recombinant frequency was compared with nucleotide distance in crosses involving markers in either the P_RM or the cy region of phage λ. For each pair of markers, we performed reciprocal four-factor crosses of the following types: (I) A⁺m₁ ⁺m₂^-B^- x A^-m₁ ^-m₂⁺B⁺; and (II) A⁺m₁ ^-m₂⁺B^- x A^-m₁ ⁺m₂^-B⁺. In crosses of type I, the frequency of A⁺m₁ ⁺m₂⁺B⁺ recombinants among total (selected) A⁺B⁺ progeny was directly proportional to nucleotide distance between m₁ and m₂ in the range from 3 to 160 nucleotides. When less than three nucleotides separated m₁ and m₂, the measured yields of m₁⁺m₂⁺ recombinants were significantly depressed.

We also found that the frequency of A⁺m₁ ⁺m₂⁺B⁺ recombinants among total A⁺B⁺ progeny was significantly lower (about 10-fold on the average) in crosses of type II than in the corresponding crosses of type I. Since mismatch correction should yield A⁺m₁ ⁺m₂⁺B⁺ recombinants with approximately equal frequencies in type I and II crosses, we suggest: (1) that most m₁⁺m₂⁺ recombinants produced in type I crosses must arise from the formation of heteroduplex structures with a discontinuity (in the source of genetic information) between sites m₁ and m₂, and (2) that mismatch correction is not a major pathway for production of recombinants for close markers in normal λ infection.

     

Endopeptidase Activity of Phage λ-Endolysin

JACOB and Fuerst^1,2 demonstrated the presence of a bacteriolytic enzyme (λ-endolysin) in the induced cultures of lysogenic Escherichia coli K12 (λ). The enzyme was later identified as the product of gene R; of phage λ³ which is involved in bacterial lysis at the end of a latent period. The enzyme is apt to form spheroplast-like structures in E. coli² and one would therefore expect its substrate to be murein.     

Structural and Functional Characterization of the Redβ Recombinase from Bacteriophage λ

The Red system of bacteriophage λ is responsible for the genetic rearrangements that contribute to its rapid evolution and has been successfully harnessed as a research tool for genome manipulation. The key recombination component is Redβ, a ring-shaped protein that facilitates annealing of complementary DNA strands. Redβ shares functional similarities with the human Rad52 single-stranded DNA (ssDNA) annealing protein although their evolutionary relatedness is not well established. Alignment of Rad52 and Redβ sequences shows an overall low level of homology, with 15% identity in the N-terminal core domains as well as important similarities with the Rad52 homolog Sak from phage ul36. Key conserved residues were chosen for mutagenesis and their impact on oligomer formation, ssDNA binding and annealing was probed. Two conserved regions were identified as sites important for binding ssDNA; a surface basic cluster and an intersubunit hydrophobic patch, consistent with findings for Rad52. Surprisingly, mutation of Redβ residues in the basic cluster that in Rad52 are involved in ssDNA binding disrupted both oligomer formation and ssDNA binding. Mutations in the equivalent of the intersubunit hydrophobic patch in Rad52 did not affect Redβ oligomerization but did impair DNA binding and annealing. We also identified a single amino acid substitution which had little effect on oligomerization and DNA binding but which inhibited DNA annealing, indicating that these two functions of Redβ can be separated. Taken together, the results provide fresh insights into the structural basis for Redβ function and the important role of quaternary structure.     

Role of the tof gene in the production and perpetuation of the λdv plasmid

Molecular Genetics and Genomics - A series of λ derivatives carrying tof mutations were tested for their ability to give rise to plasmid λ dv. Phages carrying tof mutations that distorted...     

Interaction between the Sbcc Gene of Escherichia Coli and the Gam Gene of Phage λ

gam mutants of phage lambda carrying long palindromes fail to form plaques on wild-type Escherichia coli but do grow on strains that are mutant in the sbcC gene. gam + lambda carrying the same palindrome grow on both hosts and on a host deleted for the recB, C and D genes. These results suggest that the Gam protein of lambda, known to interact also with E. coli's recBCD protein, can interact with the product of the sbcC gene.     

Complementation and characterization of the nested Rz and Rz1 reading frames in the genome of bacteriophage λ

Transposon insertions in the Rz gene of bacteriophage λ block lysis if the medium contains divalent cations at concentrations greater than 5?mM, but otherwise cause no change in phenotype. The Rz protein is thought to have an endopeptidase activity, previously reported in λ lysates, which might be involved in cleavage of oligopeptide crosslinks between glycosidic strands in the peptidoglycan and the Lpp lipoproteins of the outer bacterial membrane. Recently, a small lipoprotein has been reported as the product of a short reading frame, designated Rz1, in the +1 register within Rz. This protein has been detected in membranes of induced λ lysogens. To determine whether Rz1 has a function in the λ vegetative cycle, amber nonsense alleles of Rz and Rz1 have been constructed by site-directed mutagenesis and used for complementation and suppression analysis. Both Rzam and Rz1am alleles have phenotypes identical to those of the original Rz insertion alleles, and complement and are fully suppressed in a supE host, indicating that the two genes are independent, trans-acting genes encoding proteins required for lysis in the presence of cations. Moreover, supF suppresses Rzam but not the Rz1am mutation, and the defective Rz1am product in the supF host shows a partially dominant character and significantly retards lysis even in the absence of additional cations in the medium. Rz and Rz1 represent a unique example of two genes located in different reading frames in the same nucleotide sequence, which encode different proteins that are both required in the same physiological pathway.     

Preparation of Plasmid λdv from Bacteriophage λ: Role of Promoter-Operator in the Plasmid Replicon

A technique has been described for selection of bacteria carrying plasmid lambdadv. With this technique, the effects of mutations in the promoter-operators were compared on the production and perpetuation of the plasmid. It was found that "left" promoter-operator that controls leftward gene expressions can be deleted from the plasmid genome. Some mutations of "right" promoter-operator (pRoR) that controls expression of genes tof, O, and P affect the stability of the plasmid. However, the plasmid genome accomodates a variety of pRoR mutations within a reasonable but different degree of constitutivity. Some new promoter mutations that allow bypass of the pRoR cannot be carried in the plasmid genome. From these findings it was proposed that the plasmid replicon has one indispensable promoter-operator that controls expression of all the genes related to its own replication, although a variety of constitutive mutations can be accommodated in the pRorR.     

Tests of the Double-Strand-Break Repair Model for Red-Mediated Recombination of Phage λ and Plasmid λdv

The double-strand-break repair (DSBR) model was formulated to account for various aspects of yeast mitotic and meiotic recombination. In this study three features of the DSBR model are tested for Red-mediated recombination between phage lambda and lambda dv, a plasmid that is perfectly homologous to about 10% of lambda. The results support the applicability of the DSBR model to lambda's Red system: (1) Creating a double-strand-break (DSB) within the region of homology shared by phage and plasmid increases their genetic interaction by about 20-fold. A DSB outside the region of shared homology has no such effect. (2) Both patches, i.e., simple marker rescue, and splices, i.e., co-integration of the phage and plasmid, are stimulated by a DSB in the region of shared homology. (3) Co-integrants harbor a duplication of the region of shared homology. Among co-integrants that were formed by the creation of a DSB, there is a preferential loss of whichever allele was in cis to a utilized cut site. The DSBR model as originally formulated involves the isomerization and cleavage of Holliday junctions to resolve the canonical intermediate. We propose as an alternative mechanism that a topoisomerase can resolve the canonical DSBR intermediate.