Bacteriophage lambda DNA packaging: A mutant terminase that is independent of integration host factor

Summary ⁺ is able to grow in Escherichia coli cells lacking integration host factor (IHF), producing a burst of approximately 25% that produced in IHF⁺ cells. In vitro, however, we find that the DNA packaging enzyme terminase is strongly dependent on IHF in both cos cleavage reactions and DNA packaging reactions. The cos59 mutation renders dependent on IHF in vivo. The cos59 mutation is a deletion of 3 base pairs at the XmnI site in the cohesive end site (cos) of . Variants of cos59 that were able to grow in the absence of IHF were isolated and found to carry a mutation, called ms1, in the Nu1 gene, which codes for the small subunit of terminase. The Nu1ms1 mutation results in a change of the 40th amino acid of the Nu1 gene product from leucine to phenylalanine. The Nu1ms1 terminase was independent of IHF in packaging reactions in vitro. The results indicate that the mutation either renders terminase: (1) able to utilize some host protein other than IHF, or (2) totally independent of host factors.     

Illegitimate recombination mediated by T4 DNA topoisomerase in vitro

Summary Illegitimate recombination dependent on T4 DNA topoisomerase in a cell-free system has recently been described. In that work, recombinants between two phage DNA molecules were produced by the topoisomerase alone, without an Escherichia coli extract. In this paper, it is shown that recombination between phage and circular plasmid DNA molecules can also be detected in the presence or absence of an E. coli extract but at frequencies two or three orders of magnitude lower than that observed in the phage-phage cross. The frequency is probably lower because multiple recombination is required in the case of the phage-plasmid cross.     

Lethal effect of lambda DNA terminase in recombination deficient Escherichia coli

Summary DNA terminase is the enzyme that catalyses the cleavage of DNA concatemers into genome-size molecules and packages them into the capsid. The cleavage (DNA maturation) takes place in a specific site in the phage DNA called cos. Either one of two Escherichia coli proteins, integration host factor (IHF) and terminase host factor (THF), is required, in addition to terminase, for maturation of wild-type DNA in vitro. In vivo, at least some cos cleavage is known to occur in mutants that are unable to synthesize active IHF. No THF-defective mutants have yet been isolated. In order to determine if IHF, THF or any other host protein is involved in DNA maturation in vivo, I devised a selection for host mutants that are unable to support cos cleavage. The selection is based on the assumption that DNA terminase will kill cells by cleaving chromosomally located cos sites. I found that DNA terminase will indeed kill cells provided that they contain a chromosomal cos site and provided also that they are defective in the host recA or recB genes. These two genes are required for certain pathways of genetic recombination and repair of damaged DNA, and I suggest that they prevent terminase-induced killing by repairing broken chromosomes. Interstingly, mutation in a related host gene, recD, did not render cells susceptible to terminase killing. recD and recB both encode subunits of exonuclease V, but recD mutants, unlike recB, remain proficient in genetic recombination and repair. I found mutants that survived the lethal effect of terminase in cos-containing E. coli recA at a frequency of about 5×10^-5. About 90% of these survivors were defective in terminase synthesis, and the rest were defective in IHF function. This result suggests that in the absence of IHF in vivo cos cleavage decreases to a level that permits repair of the damage, and therefore survival, even in recombination deficient cells. The absence of mutations in any other host gene suggests that IHF is the major accessory factor in DNA maturation in vivo. Alternatively, or in addition, mutations in other accessory factors are lethal.Abbreviations gp gene product: e.g. gpA, product of gene A - () prophage state - [] plasmid-carrier state     

An efficient method for preparing high quality DNA from plant DNA libraries in lambda phage

An efficient method has been developed to improve preparation of phage particles by ammonium sulfate precipitation and to yield high quality DNA. The method, that has been used to screen plant DNA libraries constructed in vectors, is inexpensive, does not require purification of phage particles, and can be used from either plate stocks or liquid lysates. Up to 1100 g DNA was produced from 5 ml lysate obtained from agar plates.     

Alleviation of type I restriction in adenine methylase (dam) mutants of Escherichia coli

Summary The host-controlled EcoK-restriction of unmodified phage .O is alleviated in dam mutants of Escherichia coli by 100- to 300-fold. In addition, the EcoK modification activity is substantially decreased in dam ^- strains. We show that type I restriction (EcoB, EcoD and EcoK) is detectably alleviated in dam mutants. However, no relief of EcoRI restriction (Type II) occurs in dam ^- strains and only a slight effect of dam mutation on EcoP1 restriction (Type III) is observed. We interpret the alleviation of the type I restriction in dam ^- strains to be a consequence of induction of the function which interferes with type I restriction systems.     

Helical packaging of semiflexible polymers in bacteriophages

We investigate multilayered helical packaging of double-stranded DNA, or of a general polymer chain with persistence length l_b, into an ideal, inert cylindrical container, reaching densities slightly below close packaging. We calculate the free energy as a function of the packaged length, based on the energies for bending, twisting, the suffered entropy loss, and the electrostatic energy in a Debye–Hückel model. In the absence of charges on the packaged polymer, a critical packaging force can be determined, similar to the mechanism involved in DNA unzipping models. When charges are taken into consideration, in the final packaging state the charges which are chemically distant become geometrically close, and therefore a steep rise is seen in the free energy. We argue that due to the extremely ordered and almost closely packaged final state the actual packaging geometry does not influence the behaviour of the free energy, pointing towards a certain universality of this state of the polymer. Our findings are compared to a recent simulations study, showing that the model is sensitive to the screening length.     

In vivo effect of DNA repair on the transition frequency produced from a single O6-methyl- or O6-n-butyl-guanine in a T:G base pair

Summary We have previously reported some effects of DNA repair on the transition frequencies produced by an O⁶-methyl-guanine (MeG) or an O⁶-n-butyl-guanine (BuG) paired with C at the first position of the third codon in gene G of bacteriophage X174 form I'DNA (Chambers et al. 1985). We now report experiments in which the transition is produced from T:MeG or T:BuG, instead of C:MeG or C:BuG, located at this site. The site-modified DNAs were transfected into cells with normal DNA repair as well as into cells with repair defects (uvrA, uvrB, uvrC, recA, uvrArecA). The lysates were screened for phage carrying the expected transition using a characteristic change in phenotype. The data demonstrate that the transition frequency from T:BuG is low (0.3% of total phage progeny) in cells with normal repair (Escherichia coli AB1157) and increases 7-fold in uvrA cells (E. coli AB1886). A similar increase is seen in uvrB and uvrC cells (AB1885, AB1884). These data, like our previous data, indicate BuG is repaired primarily by excision. In contranst to this, the transition frequency from T:MeG is high (5±2%) in cells with normal repair. After induction of alkyl transfer repair in E. coli AB1157, the transition frequency goes up 5-fold. Compared with cells with normal repair, the transition frequency goes up 2-fold in uvrA, uvrB and uvrC cells; it goes up 1.5-fold in recA cells (E. coli AB2463). The data reinforce our earlier conclusion that MeG is repaired primarily by alkyl transfer, but the ABC excinuclease as well as RecA protein inhibit this repair process. Using the BuG data reported here and in our previous paper, we calculate that BuG pairs with a thymine residue 0.5%–0.62% of the time during replication in vivo, and that BuG markedly inhibits replication of the strand that contains it. Because of the complication introduced by alkyl transfer repair, similar calculations for MeG cannot be made from the current data.Abbreviations MeG and BuG O⁶-methyl-or O⁶-n-butyl-guanine moiety in X DNA (in each case, the plus strand nucleotide is specified first) - form I'DNA relaxed, covalently closed, circular, double-stranded DNA - Wt wild-type phenotype - Am amber phenotype - pfu plaque forming units - MNNG N-methyl-N'-nitro-N-nitrosoguanidine X mutants are named by designating the gene, the type of mutation (e.g. ms=missense), the codon number, the mutant codon and the new amino acid (where pertinent) in that order (e.g. XGam3) carries an amber in the third codon of gene G, and should not be confused with the classical am3 mutant used in the older literature to designate what is now known to be XEam7     

Lambda red-mediated synthesis of plasmid linear multimers in Escherichia coli K12

Summary Expression of the red ⁺ and gam ⁺ genes of bacteriophage in plasmids cloned in Escherichia coli wild-type cells leads to plasmid linear multimer (PLM) formation. In mutants that lack exonuclease I (sbcB sbcC), either of these functions mediates PLM formation. In order to determine whether PLM formation in sbcB sbcC mutants occurs by conservative (break-join) recombination of circular plasmids or by de novo DNA synthesis, thyA sbcB sbcC mutants were transferred from thymine- to 5-bromo-2-deoxyuridine (BUDR)-supplemented medium, concurrently with induction of red ⁺ or gam ⁺ expression, and the density distribution of plasmid molecular species was analyzed. After a period of less than one generation in the BUDR-supplemented medium, most PLM were of heavy/heavy density. Circular plasmids, as well as chromosomal DNA, were of light/light or light/heavy density. These results indicate that Red or Gam activities mediate de novo synthesis of PLM in sbcB sbcC mutants. Examination of plasmid DNA preparations from sbcB sbcC mutants expressing gam ⁺ or red ⁺ reveals the presence of two molecular species that may represent intermediates in the PLM biosynthesis pathway: single-branched circles (-structures) and PLM with single-stranded DNA tails. While Gam-mediated PLM synthesis in sbcB mutants depends on the activity of the RecF pathway genes, Red-mediated PLM synthesis, like Red-mediated recombination, is independent of recA and recF activities. One of the red ⁺ products, protein, suppresses RecA deficiency in plasmid recombination and PLM synthesis in RecBCD^– Exol^– cells. The dependence of PLM synthesis on the RecE, RecF or Red recombination pathways and the dependence of plasmid recombination by these pathways on activities that are required for plasmid replication support the proposal that PLM synthesis and recombination by these pathways are mutually dependent. We propose the hypothesis that DNA double-stranded ends, which are produced in the process of PLM synthesis, are involved in plasmid recombination by the RecE, RecF and Red pathways. Conversely, recombination-dependent priming of DNA synthesis at 3 singles-tranded DNA ends is hypothesized to initiate PLM synthesis on circular plasmid DNA templates.Abbreviations PLM plasmid linear multimers - BUDR 5-bromo-2-deoxyuridine - bp base pair     

Interactions between esterified whey proteins (alpha-lactalbumin and beta-lactoglobulin) and DNA studied by differential spectroscopy

Spectroscopic study of interactions between esterified whey proteins and nucleic acids, at neutral pH, showed positive differential spectra over a range of wavelength between 210 and 340 nm. In contrast, native forms of whey proteins added to DNA did not produce any differential spectra. The positive difference in UV absorption was observed after addition of amounts of proteins as low as 138 molar ratio (MR) of protein/DNA, indicating high sensitivity of the applied method to detect interactions between basic proteins and DNA. UV-absorption differences increased with MR of added whey protein up to saturation. The saturation points were reached at relatively lower MR in the case of methylated forms of the esterified protein as compared to its ethylated form. Saturation of nucleic acid (2996 bp long) was achieved using 850 and 1100 MR of methylated -lactoglobulin and of methylated -lactalbumin, respectively. Saturation with ethylated forms of the proteins was reached at MR of 3160 and 2750. Lysozyme, a native basic protein, showed a behavior similar to what was observed in the case of methylated forms of the dairy proteins studied. However, in the case of lysozyme, saturation was achieved at relatively lower MR (700). Methylated -casein failed to give positive spectra at pH 7 in the presence of DNA. It interacted with DNA only when the pH of the medium was lowered to 6.5, below its pI. Generally, amounts of proteins needed to saturate nucleic acid were much higher than those needed to neutralize it only electrostatically, demonstrating the presence on DNA of protein-binding sites other than the negative charges on the sugar-phosphate DNA backbones. Addition of 0.1% SDS to the medium suppressed totally all spectral differences between 210–340 nm. The presence of 5 M urea in the medium reduced only the spectral differences between 210–340 nm, pointing to the role played by hydrophobic interactions. Peptic hydrolysates of esterified and native proteins or their cationic fractions (pH > 7) produced negative differential spectra when mixed with DNA. The negative differences in UV absorption spectra were the most important in the case of peptic hydrolysates of methylated derivatives of whey proteins.     

Amelanchier alnifolia vegetation in eastern Idaho,USA and its environmental relationships

Fifteen shrub stands of Amelanchier alnifolia vegetation were studied using Braun-Blanquet procedures, tabulated, and ordered by Twinspan, Decorana, and field experience into 5 groups of stands. The stands occurred on the lower slopes of the southwestern Teton and southeastern Big Hole mountains of eastern Idaho and in-and outside a U.S. Forest Service exclosure above the Hoback River in adjacent Wyoming. The ecology of this Amelanchier vegetation as a whole and of the 5 groups of stands is discussed using Jenny's factors of soil formation (1941, 1958, 1980) as a framework. Plant indicator values (Ellenberg 1979, 1988) which rank species on their occurrences in relation to light, temperature, continentality of climate, water availability, soil reaction (pH), and soil nitrogen supply, were independently assigned to the 121 species of vascular plants encountered in the described stands.     

Endogenous interferon α/β produced by Kupffer cells inhibits interleukin-1, tumor necrosis factor α production and interleukin-2-induced activation of nonparenchymal liver cells

Summary We have previously reported liver-specific interferon (IFN) / production by murine Kupffer cells that was not observed with other tissue macrophages incubated in the absence of stimulators such as IFN or lipopolysaccharide (LPS). Consequently, while interleukin-2 (IL-2) alone induced pronounced lymphokine-activated killer (LAK) activity from splenocytes, combination of anti-IFN/ antibody with IL-2 was required to generate significant LAK activity from nonparenchymal liver cells. This endogenous IFN/ production by Kupffer cells was not induced by LPS because (a) addition of polymyxin B did not abolish the positive effects of anti-IFN/ antibody on nonparenchymal liver cells, and (b) similar results were obtained when comparing the responses of LPS-responsive C3HeB/FeJ and LPS-hyporesponsive C3H/HeJ mice. The possibility of hepatotropic infection was also ruled out in that anti-IFN/ antibody enhanced hepatic but not splenic LAK cell induction in vitro in both conventional and germfree C3H/HeN mice. IFN/ played an autoregulatory role by down-regulating the production of IL-1 and tumor necrosis factor by Kupffer cells. However, the augmenting effect of anti-IFN/ antibody on LAK induction from non-parenchymal liver cells was not mediated through an increase in the level of either IL-1 or TNF, as specific antisera against either cytokine did not abrogate this positive effect. Finally, flow-cytometry analysis showed that IFN/ significantly diminished the expression of IL-2 receptor chain, indicating an inhibition of LAK cell generation at a relatively early stage of induction.This work is supported by NIH grant RO1-28 835 and by Medical Research Funds from the Veterans Administration     

An S1 episomal gene of maize mitochondria is expressed in male sterile and fertile plants of the S-type cytoplasm

Summary Maize mitochondria of cytoplasmic male sterile (cms-S) plants contain two linear episomes, S1 (6397 bp) and S2 (5453 bp). S1 contains three long open reading frames URF2 (1017 bp), URF3 (2782 bp) and URF4 (768 bp). We have demonstrated that the URF3 sequence of S1 encodes a protein with an apparent molecular weight of 103 kDa which is found in cms-S but undetectable in cms-T, cms-C or normal (fertile) mitochondria. A translational fusion containing the 5 terminus of the lacZ gene and 800 bp of the 3 end of URF3 was isolated from a cms-S mitochondrial genomic library in the expression vector gt11. Polyclonal antibodies raised against the resulting fusion protein immunoprecipitated a 103 kDa polypeptide from among [³⁵S]-methionine-labeled cms-S mitochondrial proteins but not from normal mitochondrial proteins. The mitochondria of fertile F1 plants resulting from a cross between B37 cms-S and Ky21 (universal restorer) contain as much of this 103 kDa protein as is observed in sterile cms-S mitochondria. The mitochondria of fertile cytoplasmic revertants from cms-RD and cms-LM in a WF9 nuclear background also synthesized the 103 kDa protein. We conclude that the URF3 sequence of the S1 episome is expressed in vivo and that the presence of its gene product in maize mitochondria is not sufficient to confer the male sterile phenotype.     

The origin of crop weed communities composed of summer annuals

Summary Evidence for the origin and dating of crop weed communities composed of summer annuals is to be found in palaeobotanical and archaeological data. There is archaeological evidence that around the middle of the first millennium BC fields were taken into permanent cultivation, even on the poorer soils.Fertilization with lime and manure and Plaggenwirtschaft were evidently involved in this early agriculture. In this paper the idea is put forward that Plaggenwirtschaft resulted in a sudden increase in the number of weed species brought together in cornfields and that this practise led to the very first beginning of our modern weed communities.Translated into English by Drs. C. van Driel-Murray.