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1.
Lytic Replication of Coliphage Lambda in Salmonella typhosa Hybrids   总被引:2,自引:0,他引:2       下载免费PDF全文
Hybrids between Escherichia coli K-12 and Salmonella typhosa which conserved a continuous K-12 chromosomal diploid segment extending from pro through ara to the strA locus were sensitive to plaque formation by wild-type λ. These partially diploid S. typhosa hybrids could be lysogenized with λ and subsequently induced to produce infectious phage particles. When the K-12 genes were segregated from a lysogenic S. typhosa hybrid, phage-productive ability was no longer detectable due to loss of a genetic region necessary for vegetative replication of λ. However, λ prophage was shown to persist in a quiescent state in the S. typhosa hybrid segregant with phage-productive ability being reactivated after replacement of the essential K-12 λ replication region. Low-frequency transduction and high-frequency transduction lysates containing the gal+ genes of S. typhosa were prepared by induction of λ-lysogenic S. typhosa hybrids indicating that the attλ site is chromosomally located in S. typhosa in close proximity to the gal locus as in E. coli K-12. After propagation in S. typhosa hybrids, λ was subject to restriction by E. coli K-12 recipients, thus establishing that S. typhosa does not perform the K-12 modification of λ deoxyribonucleic acid. Hybrids of S. typhosa, however, did not restrict λ grown previously on E. coli K-12. The K-12 genetic region required for λ phage production in S. typhosa was located within min 66 to min 72 on the genetic map of the E. coli chromosome. Transfer of an F-merogenote encompassing the 66 to 72 min E. coli chromosomal region to λ-insensitive S. typhosa hybrids enabled them to replicate wild-type λ. The λ-insensitive S. typhosa hybrid, WR4255, which blocks λ replication, can be mutagenized to yield mutant strains sensitive to λvir and λimm434. These WR4255 mutants remained insensitive to plaque formation by wild-type λ.  相似文献   

2.
We have found previously that, in contrast to the free O initiator protein of λ phage or plasmid rapidly degraded by the Escherichia coli ClpP/ClpX protease, the λO present in the replication complex (RC) is protected from proteolysis. However, in cells growing in a complete medium, a temperature shift from 30 to 43°C resulted in the decay of the λO fraction, which indicated disassembly of RC. This process occurred due to heat shock induction of the groE operon, coding for molecular chaperones of the Hsp60 system. Here we demonstrate that an increase in the cellular concentration of GroEL and GroES proteins is not in itself sufficient to cause RC disassembly. Another requirement is a DNA gyrase-mediated negative resupercoiling of λ plasmid DNA, which counteracts DNA relaxation and starts to dominate 10 min after the temperature upshift. We presume that RC dissociates from λ DNA during the negative resupercoiling, becoming susceptible to the subsequent action of GroEL/S and ClpP/ClpX proteins. In contrast to λcro+, in λcro plasmid-harboring cells, the RC reveals heat shock resistance. After temperature upshift of the λcrots plasmid-harboring cells, a Cro repressor-independent control of λ DNA replication and heat shock resistance of RC are established before the period of DNA gyrase-mediated negative supercoiling. We suggest that the tight binding of RC to λ DNA is due to interaction of RC with other DNA-bound proteins, and is related to the molecular basis of the λcro plasmid replication control.  相似文献   

3.
A method is developed for analyzing in a unified manner both uniaxial and uniform biaxial strain data obtained from nearly isotropic tissues. The formulation is a direct application of nonlinear elasticity theory pertaining to large deformations. The general relation between Eulerian stress (σ) and extension ratio (λ) in soft isotropic elastic bodies undergoing uniform deformation takes the simple form: σ = ((λ3 - 1)/λ) f(λ), where f(λ) must be determined for each material. The extension ratio may be either greater than 1.0 (uniaxial elongation), or lie between zero and 1.0 (uniform biaxial extension). Simple analytical functions for f(λ) are most readily found for each tissue by plotting all data as (λ3 - 1)/λσ vs. λ. Of those tissues investigated in this way (dog pericardium and pleura, and cat mesentery and dura), all but pleura could be adequately described by a parabola: 1/f(λ) = 1/k{[(λM - λ)(λ - λm)]/[λM - λm}. In these instances, three material constants per tissue (K, λM, λm) served to predict approximately the stresses attained during both small and large deformations, in strips and sheets alike. It was further found that the uniaxial strain asymptote (λM) was linearly related to the biaxial strain asymptote (ΛM), thus effectively reducing the number of constants by one.  相似文献   

4.
A Thermus thermophilus selector strain for production of thermostable and thermoactive α-galactosidase was constructed. For this purpose, the native α-galactosidase gene (agaT) of T. thermophilus TH125 was inactivated to prevent background activity. In our first attempt, insertional mutagenesis of agaT by using a cassette carrying a kanamycin resistance gene led to bacterial inability to utilize melibiose (α-galactoside) and galactose as sole carbohydrate sources due to a polar effect of the insertional inactivation. A Gal+ phenotype was assumed to be essential for growth on melibiose. In a Gal background, accumulation of galactose or its metabolite derivatives produced from melibiose hydrolysis could interfere with the growth of the host strain harboring recombinant α-galactosidase. Moreover, the AgaT strain had to be Kms for establishment of the plasmids containing α-galactosidase genes and the kanamycin resistance marker. Therefore, a suitable selector strain (AgaT Gal+ Kms) was generated by applying integration mutagenesis in combination with phenotypic selection. To produce heterologous α-galactosidase in T. thermophilus, the isogenes agaA and agaB of Bacillus stearothermophilus KVE36 were cloned into an Escherichia coli-Thermus shuttle vector. The region containing the E. coli plasmid sequence (pUC-derived vector) was deleted before transformation of T. thermophilus with the recombinant plasmids. As a result, transformation efficiency and plasmid stability were improved. However, growth on minimal agar medium containing melibiose was achieved only following random selection of the clones carrying a plasmid-based mutation that had promoted a higher copy number and greater stability of the plasmid.  相似文献   

5.
The lon mutants of Escherichia coli grow apparently normally except that, after temporary periods of inhibition of deoxyribonucleic acid synthesis, septum formation is specifically inhibited. Under these conditions, long, multinucleate, nonseptate filaments result. The lon mutation also creates a defect such that wild-type bacteriophage λ fails to lysogenize lon mutants efficiently and consequently forms clear plaques on a lon host. Two lines of evidence suggest that this failure probably results from interference with expression of the λcI gene, which codes for repressor, or with repressor action:-(i) when a lon mutant was infected with a λcII, cIII, or c Y mutant, there was an additive effect between the lon mutation and the λc mutations upon reduction of lysogenization frequency; and (ii) lon mutants permitted the growth of the λcro mutant under conditions in which the repressor was active. The isolation of λ mutants (λtp) which gained the ability to form turbid plaques on lon cells is also reported.  相似文献   

6.
Ronald D. Porter 《Genetics》1983,105(2):247-257
Several aspects of the recombination resulting from λ plac5 transduction were investigated in strains of Escherichia coli K-12 that use the RecE or RecF recombination pathways. In a RecBC pathway strain, F42lac recombination with λplac5 is 20- to 50-fold higher than chromosomal lac times λplac5 recombination, and this recombination enhancement is largely dependent on constitutive expression of F42lac fertility functions. Here, it was observed that F42 lac fertility functions do not effect the ability of F42lac to recombine with λplac5 in a RecE or RecF pathway strain. Therefore, the enhancement observed in a Rec+ (or RecBC pathway) strain is directly dependent on the recBC gene product. The end product of recombination between λplac5 and either F42lac or chromosomal lac in RecE and RecF pathway strains was monitored by scoring for addition and substitution transductants. It was observed that the percentage of addition transductants was lower in all cases for RecE and RecF pathway strains as compared with RecBC pathway or a recB strain. It is concluded that the introduction of sbcA or sbcB into a recB strain produces a change in recombination mechanism that is reflected in the nature of the end product of recombination.  相似文献   

7.
DNA polymerase λ (pol λ) is a member of the X family DNA polymerases and is endowed with multiple enzymatic activities. In this work we investigated the in vitro miscoding properties of full-length, human pol λ either in the absence or in the presence of the human auxiliary proteins proliferating cell nuclear antigen (PCNA) and replication protein A (RP-A). Our data suggested that (i) pol λ had an intrinsic ability to create mismatches and to incorporate ribonucleotides at nearly physiological Mn++ and Mg++ concentrations; (ii) the sequence of the template-primer could influence the misincorporation frequency of pol λ; (iii) pol λ preferentially generated G:T and G:G mismatches; (iv) RP-A, but not PCNA, selectively prevented misincorporation of an incorrect nucleotide by pol λ, without affecting correct incorporation and (v) this inhibitory effect required a precise ratio between the concentrations of pol λ and RP-A. Possible physiological implications of these findings for the in vivo fidelity of pol λ are discussed.  相似文献   

8.
Decay-associated fluorescence spectra of the green alga Scenedesmus obliquus have been measured by single-photon timing with picosecond resolution in various states of light adaptation. The data have been analyzed by applying a global data analysis procedure. The amplitudes of the decay-associated spectra allow a determination of the relative antenna sizes of the photosystems. We arrive at the following conclusions: (a) The fluorescence kinetics of algal cells with open PS II centers (F0 level) have to be described by a sum of three exponential components. These decay components are attributed to photosystem (PS) I (τ ≈ 85 ps, λmaxem ≈ 695-700 nm), open PS II α-centers (τ ≈ 300 ps, λmaxem = 685 nm), and open PS II β-centers (τ ≈ 600 ps, λmaxem = 685 nm). A fourth component of very low amplitude (τ ≈ 2.2-2.3 ns, λmaxem = 685 nm) derives from dead chlorophyll. (b) At the Fmax level of fluorescence there are also three decay components. They originate from PS I with properties identical to those at the F0 level, from closed PS II α-centers (τ ≈ 2.2 ns, λmaxem = 685 nm) and from closed PS β-centers (τ ≈ 1.2 ns, λmaxem = 685 nm). (c) The major effect of light-induced state transitions on the fluorescence kinetics involves a change in the relative antenna size of α- and β-units brought about by the reversible migration of light-harvesting complexes between α-centers and β-centers. (d) A transition to state II does not measurably increase the direct absorption cross-section (antenna size) of PS I. Our data can be rationalized in terms of a model of the antenna organization that relates the effects of state transitions and light-harvesting complex phosphorylation with the concepts of PS II α,β-heterogeneity. We discuss why our results are in disagreement with those of a recent lifetime study of Chlorella by M. Hodges and I. Moya (1986, Biochim. Biophys. Acta., 849:193-202).  相似文献   

9.
β-D-galactofuranose (Galf) is a component of polysaccharides and glycoconjugates and its transferase has been well analyzed. However, no β-D-galactofuranosidase (Galf-ase) gene has been identified in any organism. To search for a Galf-ase gene we screened soil samples and discovered a strain, identified as a Streptomyces species by the 16S ribosomal RNA gene analysis, that exhibits Galf-ase activity for 4-nitrophenyl β-D-galactofuranoside (pNP-β-D-Galf) in culture supernatants. By draft genome sequencing of the strain, named JHA19, we found four candidate genes encoding Galf-ases. Using recombinant proteins expressed in Escherichia coli, we found that three out of four candidates displayed the activity of not only Galf-ase but also α-L-arabinofuranosidase (Araf-ase), whereas the other one showed only the Galf-ase activity. This novel Galf-specific hydrolase is encoded by ORF1110 and has an optimum pH of 5.5 and a Km of 4.4 mM for the substrate pNP-β-D-Galf. In addition, this enzyme was able to release galactose residue from galactomannan prepared from the filamentous fungus Aspergillus fumigatus, suggesting that natural polysaccharides could be also substrates. By the BLAST search using the amino acid sequence of ORF1110 Galf-ase, we found that there are homolog genes in both prokaryotes and eukaryotes, indicating that Galf-specific Galf-ases widely exist in microorganisms.  相似文献   

10.
11.
Although closely related at the molecular level, the capsular polysaccharide (CPS) of serotype 10F Streptococcus pneumoniae and coaggregation receptor polysaccharide (RPS) of Streptococcus oralis C104 have distinct ecological roles. CPS prevents phagocytosis of pathogenic S. pneumoniae, whereas RPS of commensal S. oralis functions as a receptor for lectin-like adhesins on other members of the dental plaque biofilm community. Results from high resolution NMR identified the recognition region of S. oralis RPS (i.e. Galfβ1–6GalNAcβ1–3Galα) in the hexasaccharide repeat of S. pneumoniae CPS10F. The failure of this polysaccharide to support fimbriae-mediated adhesion of Actinomyces naeslundii was explained by the position of Galf, which occurred as a branch in CPS10F rather than within the linear polysaccharide chain, as in RPS. Carbohydrate engineering of S. oralis RPS with wzy from S. pneumoniae attributed formation of the Galf branch in CPS10F to the linkage of adjacent repeating units through sub terminal GalNAc in Galfβ1–6GalNAcβ1–3Galα rather than through terminal Galf, as in RPS. A gene (wcrD) from serotype 10A S. pneumoniae was then used to engineer a linear surface polysaccharide in S. oralis that was identical to RPS except for the presence of a β1–3 linkage between Galf and GalNAcβ1–3Galα. This polysaccharide also failed to support adhesion of A. naeslundii, thereby establishing the essential role of β1–6-linked Galf in recognition of adjacent GalNAcβ1–3Galα in wild-type RPS. These findings, which illustrate a molecular approach for relating bacterial polysaccharide structure to function, provide insight into the possible evolution of S. oralis RPS from S. pneumoniae CPS.  相似文献   

12.
The oxygen consumption of a starved chlorophyll-free, yellow mutant of Chlorella vulgaris is enhanced by very small amounts of blue light (λ 450 mμ); a saturation level is reached at about 500 ergs cm−2 sec−1. At that intensity the respiration is about 3 times greater than in the dark. An action spectrum for the enhancement of respiration shows 2 peaks around λ 450 and 375 mμ. Flavins and cis-carotenoids are discussed as the pigments involved.  相似文献   

13.
To investigate the relationship between atomic topology, vibrational and electronic properties and superconductivity of bismuth, a 216-atom amorphous structure (a-Bi216) was computer-generated using our undermelt-quench approach. Its pair distribution function compares well with experiment. The calculated electronic and vibrational densities of states (eDOS and vDOS, respectively) show that the amorphous eDOS is about 4 times the crystalline at the Fermi energy, whereas for the vDOS the energy range of the amorphous is roughly the same as the crystalline but the shapes are quite different. A simple BCS estimate of the possible crystalline superconducting transition temperature gives an upper limit of 1.3 mK. The e-ph coupling is more preponderant in a-Bi than in crystalline bismuth (x-Bi) as indicated by the λ obtained via McMillan’s formula, λc = 0.24 and experiment λa = 2.46. Therefore with respect to x-Bi, superconductivity in a-Bi is enhanced by the higher values of λ and of eDOS at the Fermi energy.  相似文献   

14.
15.
The known archaeal family B DNA polymerases are unable to participate in the PCR in the presence of uracil. Here, we report on a novel archaeal family B DNA polymerase from Nanoarchaeum equitans that can successfully utilize deaminated bases such as uracil and hypoxanthine and on its application to PCR. N. equitans family B DNA polymerase (Neq DNA polymerase) produced λ DNA fragments up to 10 kb with an approximately 2.2-fold-lower error rate (5.53 × 10−6) than Taq DNA polymerase (11.98 × 10−6). Uniquely, Neq DNA polymerase also amplified λ DNA fragments using dUTP (in place of dTTP) or dITP (partially replaced with dGTP). To increase PCR efficiency, Taq and Neq DNA polymerases were mixed in different ratios; a ratio of 10:1 efficiently facilitated long PCR (20 kb). In the presence of dUTP, the PCR efficiency of the enzyme mixture was two- to threefold higher than that of either Taq and Neq DNA polymerase alone. These results suggest that Neq DNA polymerase and Neq plus DNA polymerase (a mixture of Taq and Neq DNA polymerases) are useful in DNA amplification and PCR-based applications, particularly in clinical diagnoses using uracil-DNA glycosylase.  相似文献   

16.
Recent work of Sottoriva, Graham, and collaborators have led to the controversial claim that exponentially growing tumors have a site frequency spectrum that follows the 1/f law consistent with neutral evolution. This conclusion has been criticized based on data quality issues, statistical considerations, and simulation results. Here, we use rigorous mathematical arguments to investigate the site frequency spectrum in the two-type model of clonal evolution. If the fitnesses of the two types are λ0 < λ1, then the site frequency spectrum is c/fα where α = λ01. This is due to the advantageous mutations that produce the founders of the type 1 population. Mutations within the growing type 0 and type 1 populations follow the 1/f law. Our results show that, in contrast to published criticisms, neutral evolution in an exponentially growing tumor can be distinguished from the two-type model using the site frequency spectrum.  相似文献   

17.
Prophages are ubiquitous elements within bacterial chromosomes and affect host physiology and ecology in multiple ways. We have previously demonstrated that phage-induced lysis is required for extracellular DNA (eDNA) release and normal biofilm formation in Shewanella oneidensis MR-1. Here, we investigated the regulatory mechanisms of prophage λSo spatiotemporal induction in biofilms. To this end, we used a functional fluorescence fusion to monitor λSo activation in various mutant backgrounds and in response to different physiological conditions. λSo induction occurred mainly in a subpopulation of filamentous cells in a strictly RecA-dependent manner, implicating oxidative stress-induced DNA damage as the major trigger. Accordingly, mutants affected in the oxidative stress response (ΔoxyR) or iron homeostasis (Δfur) displayed drastically increased levels of phage induction and abnormal biofilm formation, while planktonic cells were not or only marginally affected. To further investigate the role of oxidative stress, we performed a mutant screen and identified two independent amino acid substitutions in OxyR (T104N and L197P) that suppress induction of λSo by hydrogen peroxide (H2O2). However, λSo induction was not suppressed in biofilms formed by both mutants, suggesting a minor role of intracellular H2O2 in this process. In contrast, addition of iron to biofilms strongly enhanced λSo induction and eDNA release, while both processes were significantly suppressed at low iron levels, strongly indicating that iron is the limiting factor. We conclude that uptake of iron during biofilm formation triggers λSo-mediated lysis of a subpopulation of cells, likely by an increase in iron-mediated DNA damage sensed by RecA.  相似文献   

18.
An increasing number of independent studies indicate that the atypical protein kinase C (PKC) isoforms (aPKCs) are critically involved in the control of cell proliferation and survival. The aPKCs are targets of important lipid mediators such as ceramide and the products of the PI 3-kinase. In addition, the aPKCs have been shown to interact with Ras and with two novel proteins, LIP (lambda-interacting protein; a selective activator of λ/ιPKC) and the product of par-4 (a gene induced during apoptosis), which is an inhibitor of both λ/ιPKC and ζPKC. LIP and Par-4 interact with the zinc finger domain of the aPKCs where the lipid mediators have been shown to bind. Here we report the identification of p62, a previously described phosphotyrosine-independent p56lck SH2-interacting protein, as a molecule that interacts potently with the V1 domain of λ/ιPKC and, albeit with lower affinity, with ζPKC. We also show in this study that ectopically expressed p62 colocalizes perfectly with both λ/ιPKC and ζPKC. Interestingly, the endogenous p62, like the ectopically expressed protein, displays a punctate vesicular pattern and clearly colocalizes with endogenous λ/ιPKC and endogenous ζPKC. P62 colocalizes with Rab7 and partially with lamp-1 and limp-II as well as with the epidermal growth factor (EGF) receptor in activated cells, but not with Rab5 or the transferrin receptor. Of functional relevance, expression of dominant negative λ/ιPKC, but not of the wild-type enzyme, severely impairs the endocytic membrane transport of the EGF receptor with no effect on the transferrin receptor. These findings strongly suggest that the aPKCs are anchored by p62 in the lysosome-targeted endosomal compartment, which seems critical for the control of the growth factor receptor trafficking. This is particularly relevant in light of the role played by the aPKCs in mitogenic cell signaling events.  相似文献   

19.
Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 Cλ genes, each preceded by a Jλ gene, and 86 potentially functional Vλ genes upstream of (Jλ-Cλ)n. The Igκ locus contains a single Cκ gene, 6 Jκs and 62 functional Vκs. All VL genes are classified into a total of 31 families: 19 Vλ families and 12 Vκ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed Vλ and Vκ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates.  相似文献   

20.

Purpose

To develop a diffusion-tensor-imaging (DTI) protocol that is sensitive to the complex diffusion and perfusion properties of the healthy and malignant pancreas tissues.

Materials and Methods

Twenty-eight healthy volunteers and nine patients with pancreatic-ductal-adenocacinoma (PDAC), were scanned at 3T with T2-weighted and DTI sequences. Healthy volunteers were also scanned with multi-b diffusion-weighted-imaging (DWI), whereas a standard clinical protocol complemented the PDAC patients’ scans. Image processing at pixel resolution yielded parametric maps of three directional diffusion coefficients λ1, λ2, λ3, apparent diffusion coefficient (ADC), and fractional anisotropy (FA), as well as a λ1-vector map, and a main diffusion-direction map.

Results

DTI measurements of healthy pancreatic tissue at b-values 0,500 s/mm2yielded: λ1 = (2.65±0.35)×10−3, λ2 = (1.87±0.22)×10−3, λ3 = (1.20±0.18)×10−3, ADC = (1.91±0.22)×10−3 (all in mm2/s units) and FA = 0.38±0.06. Using b-values of 100,500 s/mm2 led to a significant reduction in λ1, λ2, λ3 and ADC (p<.0001) and a significant increase (p<0.0001) in FA. The reduction in the diffusion coefficients suggested a contribution of a fast intra-voxel-incoherent-motion (IVIM) component at b≤100 s/mm2, which was confirmed by the multi-b DWI results. In PDACs, λ1, λ2, λ3 and ADC in both 0,500 s/mm2 and 100,500 s/mm2 b-values sets, as well as the reduction in these diffusion coefficients between the two sets, were significantly lower in comparison to the distal normal pancreatic tissue, suggesting higher cellularity and diminution of the fast-IVIM component in the cancer tissue.

Conclusion

DTI using two reference b-values 0 and 100 s/mm2 enabled characterization of the water diffusion and anisotropy of the healthy pancreas, taking into account a contribution of IVIM. The reduction in the diffusion coefficients of PDAC, as compared to normal pancreatic tissue, and the smaller change in these coefficients in PDAC when the reference b-value was modified from 0 to 100 s/mm2, helped identifying the presence of malignancy.  相似文献   

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