Electron microscopy study of viral DNA packaging with lambda head mutants

In a previous study, various intermediates in λ DNA packaging were visualized after lysis of λ-infected cells with osmotic shock and sedimentation through a sucrose formalin cushion onto electron microscope grids. Along this line, a systematic screening for intermediates accumulated in all head mutants available was performed. λA^?-infected cells accumulate only empty spherical protein shells (petit λ) bound at an intermediate point along the DNA thread. In situ digestion experiments with restriction endonuclease EcoRI show that the petit λ-DNA complexes are formed at a fixed point on the DNA concatemer. In $\text{λNu1}{}^{\mathrm{?}}\text{-}\text{infected}$ cells, however, most petit λ was not bound to DNA. In Fec^? cells, which are defective in formation of concatemers but normal in head protein synthesis, most petit λ did not sediment onto the carbon film of the grid. In $\text{D}{}^{\mathrm{?}}$ mutant, petit λ, partially full heads and empty heads with released DNA were observed. λFI^?-infected cells also accumulate petit λ and partially full heads. The present studies suggest that protein pNu1 is required for complex formation between head precursors and DNA concatemers, $\text{p}\text{A}$ for the initiation of DNA packaging, $\text{p}\text{D}$ and $\text{p}\text{F}$I for the promotion of DNA packaging, and $\text{p}\text{D}$ for stabilization of head structures. The results obtained with other head mutants involved in formation of mature proheads and head completion confirm earlier results obtained by different techniques.     

Dimers of escherichia coli F' factors

Covalently closed circular DNA dimers of several $\text{E.}$$\text{coli}$ sex factors have been isolated. One of these, F′451, a dimer of F′450, has a molecular weight of $\text{ca.}$ 230 × 10⁶ daltons. F′451 (λ) containing a λ prophage has a molecular weight of 260 × 10⁶ and is probably the largest covalent closed circle of DNA yet reported. These dimers arise spontaneously and are of unknown origin and significance.     

Terminal fragments of herpes simplex virus DNA produced by restriction endonuclease.

Digestion of HSV-1 DNA with λ 5′-exonuclease prior to digesting the DNA with the $\text{Eco R}$ I restriction endonuclease specifically affects two of the fragments normally obtained after restriction endonuclease digestion. Therefore these two fragments contain the sequences which occur at the termini of HSV-1 DNA. One of the fragments affected is a “minor” fragment which is always present in less than molar yield. The possible relationship between the occurrence of minor $\text{Eco R}$ I fragments and the partial refractoriness of HSV-1 DNA to λ 5′-exonuclease digestion is discussed.     

Synthesis and decay of lambda DNA replication proteins in minicells

The coliphage λ DNA replication proteins, the $\text{O}$- and $\text{P}$-gene products, have been identified by infection of nonpermissive $\text{Escherichia coli}$ minicells with the appropriate λ amber mutants as proteins of a molecular weight of about 34000 and 23000, respectively. Proteins of exactly the same size were found in minicells harbouring the plasmid $\text{λ}\text{dv}$. Both proteins seem to be synthesized at the same rate. In λ-infected minicells, as well as in $\text{lambda;}\text{dv}$-harbouring minicells the pulse-and-chase experiments have shown an exceptionally rapid decay of the O-protein.     

Mutational alteration of Bacillus subtilis DNA polymerase 3 to hydroxyphenylazopyrimidine resistance: polymerase 3 is necessary for DNA replication

A spontaneous mutant of $\text{Bacillus}\text{subtilis}$ resistant to killing by two hydroxyphenylazopyrimidines has been isolated. The DNA polymerase III of this mutant is resistant to inhibition by these drugs. The K_i for 6-(p-hydroxyphenylazo)-uracil (HPUra) is 20 μM, about 40 times higher than the K_i of the wild-type enzyme. The mutant and wild-type polymerases behave similarly during purification, are sensitive to N-ethylmaleimide and to 0.1 M KCl, and have the same K_m for dGTP (0.5 μM). The HPUra inhibition of both enzymes is attenuated competitively by dGTP. We conclude that polymerase III is the target for hydroxyphenylazopyrimidines $\text{in}\text{vivo}$, and since the drugs specifically inhibit replicative DNA synthesis, polymerase III is necessary for DNA replication.     

The secondary attachment site for bacteriophage lambda in the proA/B gene of Escherichia coli

We have determined the nucleotide sequence of a secondary λ attachment site in $\text{pro}\text{A}\text{B}$, a site that accounts for 3% of lysogens isolated from Escherichia coli strains deleted for the primary site. Direct sequence analysis of the transducing bacteriophages carrying the left and right att junctions, as well as the recombinant pro⁺ phage reveals that the $\text{pro}\text{A}\text{B}$ site shares an 11-nucleotide interrupted homology with the core sequence of the primary site. We have compared the $\text{pro}\text{A}\text{B}$att site with other secondary attachment sites to gain insights into the structural features important for λ integration.     

Effect of aphidicolin on viral and human DNA polymerases.

DNA polymerases induced by Herpes simplex and Vaccinia viruses are inhibited by aphidicolin and this inhibition is probably the basis of its antiviral activity $\text{in vivo}$. Its possible clinical use is however hampered by the concomitant effect on human replicative DNA polymerase α. The inhibition of human α-polymerase is reversible both $\text{in}$$\text{vitro}$ and $\text{in vivo}$ and the changes in the rate of incorporation of thymidine into DNA, following treatment with aphidicolin for a generation time, indicate the likely synchronization of the cells due to this agent. DNA polymerase β, which has recently been shown to carry out repair synthesis of damaged nuclear DNA, is not inhibited by aphidicolin either $\text{in vitro}$ on $\text{in vivo}$ suggesting that the drug could allow a rapid and simple evaluation of DNA repair synthesis due to DNA polymerase β.     

Parental RF formation of phages ⊘X174 and M13 requires the dnaZ gene product of Escherichiacoli

A functional $\text{dnaZ}$ product, known to be essential for host DNA polymerization and for the growth of coliphages ?X174 and M13, is required for the $\text{in}\text{vivo}$ single strand to replicative form conversions of both of these phages.     

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.     

ATP-independent, DNA synthesis by DNA polymerase II in toluenized Bacillus subtilis

Phleomycin stimulates ATP-independent DNA repair synthesis by polymerase II in toluenized $\text{B. subtilis}$ cells. In the presence of ATP it also increases the synthesis, with BrdUTP, of DNA with a density between that of normal DNA and hybrid DNA, and it enhances replicative DNA synthesis by polymerase III.     

Genetic transformation in E.coli: The inhibitory role of the recBC DNase

Genetic transformation of $\text{E.}\text{coli}$ for various chromosomal markers was accomplished by (i) using recipient cells that lack the $\text{recBC}$ DNase but were recombination proficient due to $\text{sbcA}$ or $\text{sbcB}$ mutations and (ii) treating the recipient cells with CaCl₂ at a concentration that facilitates transfection by λ DNA. Cotransformation of three markers ($\text{thr}{}^{\mathrm{+}}\text{ara}{}^{\mathrm{+.}}\text{leu}{}^{\mathrm{+}}$) was found to depend on the molecular weight of the transforming DNA.     

Activation of the template activity of isolated rat liver nuclei for DNA synthesis and its inhibition by NAD

The template activity of isolated rat liver nuclei for DNA synthesis assayed with $\text{E.}$$\text{coli}$ DNA polymerase was found to be dependent upon the presence of Ca²⁺ or Mg²⁺ in the incubation medium. DNA was prepared from isolated nuclei subjected to conditions which activated the template and centrifuged in an alkaline sucrose gradient. The distribution profile showed that smaller fragments were formed, suggesting enhancement of endonucleolytic activity. When isolated nuclei were incubated with NAD to induce poly(adenosine diphosphate ribose) formation and were subjected to the activation conditions, the template for DNA synthesis remained unchanged. The distribution profile in an alkaline sucrose gradient of DNA prepared from these nuclei and control nuclei was identical. The present findings suggest that the template-activating system for DNA synthesis was blocked when isolated nuclei were treated with NAD $\text{in}$$\text{vitro}$.     

Preliminary crystallographic data on the human lambda III Bence Jones protein dimer Cle

A complete human λ Bence Jones protein dimer (Cle) has been isolated and crystallized. Protein Cle was characterized immunochemically and chemically as having a variable region amino acid sequence associated with light chains of the λ chain subgroup, λIII, and a constant region sequence characteristic of “non-Mcg” type λ chains. Bence Jones protein Cle contains two covalently bound intact monomers, each having a molecular weight of ~23,000. Crystals of Bence Jones protein Cle, obtained from ammonium sulfate solutions, diffract to 2.6 Å resolution and have the orthorhombic space group P2₁2₁2₁ with cell dimensions $\text{a = 113.0}\text{A}\text{?}\text{, b = 72.3}\text{A}\text{?}\text{,}\text{and}\text{c = 48.9}\text{A}\text{?}$. The asymmetric unit consists of a dimer with a molecular weight of ~ 46,000.     

Uncoupling of mitochondrial oxidative phosphorylation by thallium.

The mechanism of integration of λ$\text{bio}$ll, which is deleted of all the known λ recombination genes, was studied using $\text{bio}$ deleted hosts as recipients. The presence of $\text{rec}$BC DNase and $\text{exo}$I in the recipient cells affected the fate of λ$\text{bio}$ll DNA. In nine of ten $\text{imm}\text{λ}{}^{\mathrm{+}}$ transductants, insertion of the λ$\text{bio}$ll genome took place somewhere between J and N and the remaining one had abnormally permuted prophage λ. In this lysogen (#42), the sequence of prophage genes was similar to that of vegetative phage λ. The properties of lysogen #42 were compared with those of other lysogens.     

Interconversion of thymine and thymidine in a thymine requiring strain of Bacillus subtilis

In a $\text{B. subtilis}$ Thy^? strain, thymidine is rapidly converted into thymine and, at the steady state, the pool size of thymidine is very small as compared to that of thymine. Consequently when such strain is used for pulse incorporation experiments with labelled thymidine paradoxical results are obtained. A quantitative estimation of the rate of DNA synthesis can only be obtained by thymine pulses or by cumulative incorporation experiments. We also pr$\text{e}$ sent evidence that, during a short pulse, thymidine is mainly utilized for replicative DNA synthesis.     

Ribosomal assembly defectivity in a set of amber mutants of Escherichia coli.

Five amber mutations affecting essential genes of $\text{Escherichia coli}$ have been isolated. The procedure relies on P1-mediated localized mutagenesis(1) and on the use of a recipient strain carrying a strong but instable suppressor gene and a particular thermoinducible λ prophage which kills suppressor hosts at 42°C (2). All five mutations map close to the $\text{spcA}$ gene, in a region which codes essentially for ribosomal proteins. Strains harboring the mutations were studied biochemically; all five exhibit defective ribosomal assembly upon loss of suppression.     

Characterization of DNA from Rhizobium cells and their bacteriods from root nodules

DNA from two rhizobial strains $\text{Cicer}$ and $\text{Phaseolus}$ and their bacteroids from root nodules have been isolated, purified and characterized for thermal denaturation temperature and buoyant density. Bacteroid DNA had a lower Tm value and buoyant density comparad to $\text{Rhizobium}$ cell DNA. The calculated GC content of becteroid DNA was lower than the $\text{Rhizobium}$ cell DNA.     

Inhibition of DNA replicative synthesis and DNA repair synthesis in human and mouse cells in culture by cigarette smoke condensate fractions.

Twelve cigarette smoke condensate fractions were tested for their ability to inhibit replicative DNA synthesis and DNA excision repair synthesis in cultures of human fibroblasts and Swiss mouse embryo cells. None of the fractions showed any specificity for the inhibition of DNA repair and, in general, repair synthesis was less sensitive to inhibition than was replicative synthesis. There was some correlation between the inhibitory action of the various fractions and their activity in bioassays performed in other laboratories, including $\text{in vitro}$ cell transformation and bacterial mutagenicity. In most cases, DNA synthesis in the human cells was more sensitive to inhibition than it was in the mouse cells. The specific compounds in the condensate fractions which are responsible for their activity have not been identified.     

The argECBH-bearing EcoRI segment of the Escherichia coli chromosome

The transducing phage λd$\text{arg}\text{14}$, carrying a portion of the $\text{E. coli}$ chromosome including $\text{argECBH}$, is derived from the heat-inducible, lysis-defective strain λy199, which has the $\text{b}\text{519}$ and $\text{b}\text{515}$ deletions. Cleavage of λy199 DNA by $\text{Eco}$RI endonuclease, followed by agarose slab gel electrophoresis, results in bands corresponding to the known C, D, E, and F segments of λ, and a segment A′ (A plus B minus $\text{b}\text{519}$ minus $\text{b}\text{515}$, the cleavage site between A and B being eliminated). Cleavage of λd$\text{arg}\text{14}$ DNA by $\text{Eco}$RI yields the expected D, E, and F segments of λ and four other segments, termed 14-1 through 14-4, whose length is 17.5, 6.2, 3.0, and 2.0 kilobases, respectively, as determined by electron microscopy and corroborated by electrophoretic mobility. Heteroduplex analysis shows that the $\text{E. coli argECBH}$ cluster is on the 14-1 segment.     

DNA replication in Physarum polycephalum: characterization of replication products made in isolated nuclei

Nuclei isolated from synchronous S-phase plasmodia of the myxomycete $\text{Physarum polycephalum}$ were competent in production of low molecular weight DNA replication intermediates. Furthermore, these nuclei showed some competence in joining these fragments into DNA of intermediate molecular weight. The DNA molecules made $\text{in vitro}$ could be correlated with products made $\text{in vivo}$.