State Transitions in the Green Alga Scenedesmus Obliquus Probed by Time-Resolved Chlorophyll Fluorescence Spectroscopy and Global Data Analysis

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, λ_max^em ≈ 695-700 nm), open PS II α-centers (τ ≈ 300 ps, λ_max^em = 685 nm), and open PS II β-centers (τ ≈ 600 ps, λ_max^em = 685 nm). A fourth component of very low amplitude (τ ≈ 2.2-2.3 ns, λ_max^em = 685 nm) derives from dead chlorophyll. (b) At the F_max 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, λ_max^em = 685 nm) and from closed PS β-centers (τ ≈ 1.2 ns, λ_max^em = 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).     

Simple Uniaxial and Uniform Biaxial Deformation of Nearly Isotropic Incompressible Tissues

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{[(λ_M - λ)(λ - λ_m)]/[λ_M - λ_m}. In these instances, three material constants per tissue (K, λ_M, λ_m) 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 (λ_M) was linearly related to the biaxial strain asymptote (Λ_M), thus effectively reducing the number of constants by one.     

Molecular Mechanism of Heat Shock-Provoked Disassembly of the Coliphage λ Replication Complex

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.     

Estimation of Parameters of Rotatory Dispersion Curves of Proteins

Estimation of the constants a, b, and λ₀ by means of the standard Moffitt-Yang plot is evaluated. It is found that the method is very insensitive as an estimation procedure and that large errors in b may be expected. Expressions for the maximum-likelihood estimates of the constants are derived.     

Human replication protein A can suppress the intrinsic in vitro mutator phenotype of human DNA polymerase lambda

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.     

Deoxyribonucleic Acid Replication in λ Bacteriophage Mutants

After ultraviolet light induction of Escherichia coli K-12 strain W3350(λ), several structural intermediate forms of phage deoxyribonucleic acid (DNA) are synthesized. The early defective lysogens of λ, sus O₈, sus P₃, and T₁₁, were found to synthesize none of the DNA structural intermediates. A lysogen believed to be defective in all known phage activities, λsus N₇, was found to be able to synthesize an early phage DNA intermediate. The lysogen λsus Q₂₁, defective in late phage functions, is able to synthesize the early phage DNA intermediate and a concatenated molecule of greater molecular weight than the mature λ DNA.     

A Comprehensive Analysis of the Phylogeny,Genomic Organization and Expression of Immunoglobulin Light Chain Genes in Alligator sinensis,an Endangered Reptile Species

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 C_λ genes, each preceded by a J_λ gene, and 86 potentially functional V_λ genes upstream of (J_λ-C_λ)_n. The Igκ locus contains a single C_κ gene, 6 J_κs and 62 functional V_κs. All V_L genes are classified into a total of 31 families: 19 V_λ families and 12 V_κ 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 V_λ and V_κ. 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.     

Lytic Replication of Coliphage Lambda in Salmonella typhosa Hybrids

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.     

Interpreting scratch assays using pair density dynamics and approximate Bayesian computation

Quantifying the impact of biochemical compounds on collective cell spreading is an essential element of drug design, with various applications including developing treatments for chronic wounds and cancer. Scratch assays are a technically simple and inexpensive method used to study collective cell spreading; however, most previous interpretations of scratch assays are qualitative and do not provide estimates of the cell diffusivity, D, or the cell proliferation rate, λ. Estimating D and λ is important for investigating the efficacy of a potential treatment and provides insight into the mechanism through which the potential treatment acts. While a few methods for estimating D and λ have been proposed, these previous methods lead to point estimates of D and λ, and provide no insight into the uncertainty in these estimates. Here, we compare various types of information that can be extracted from images of a scratch assay, and quantify D and λ using discrete computational simulations and approximate Bayesian computation. We show that it is possible to robustly recover estimates of D and λ from synthetic data, as well as a new set of experimental data. For the first time, our approach also provides a method to estimate the uncertainty in our estimates of D and λ. We anticipate that our approach can be generalized to deal with more realistic experimental scenarios in which we are interested in estimating D and λ, as well as additional relevant parameters such as the strength of cell-to-cell adhesion or the strength of cell-to-substrate adhesion.     

Analysis of Mice with Single and Multiple Copies of Transgenes Reveals a Novel Arrangement for the λ5-VpreB1 Locus Control Region

The murine λ5-V_preB1 locus encodes two proteins that form part of the pre-B-cell receptor and play a key role in B-lymphocyte development. We have identified a locus control region (LCR) which is responsible for coordinate activation of both genes in pre-B cells. Analysis of mice with single and multiple copies of transgenes shows a clear difference in the expression behavior of the genes depending on the transgene copy number. While expression of both λ5 and V_preB1 in single- and two-copy integrations requires the presence of a set of DNase I hypersensitive sites located 3′ of the λ5 gene, small fragments containing the genes have LCR activity when arranged in multiple-copy tandem arrays, indicating that additional components of the LCR are located within or close to the genes. The complete LCR is capable of driving efficient copy-dependent expression of a λ5 gene in pre-B cells even when it is integrated into centomeric γ-satellite DNA. The finding that activation of expression of the locus by positively acting factors is fully dominant over the silencing effect of heterochromatin has implications for models for chromatin-mediated gene silencing during B-cell development.     

Appearance of Virulent Bacteriophages in Lysogenic E. coli Cultures after Prolonged Growth in the Presence of Triethylenemelamine

Continuous culture of coli 12λ, P22, 600-434, 600-21, and 600-299 in the presence of triethylenemelamine (TEM) results in the appearance of a new virulent virus which attacks the parent culture. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) is effective with 600-21 and ultraviolet light with 12λ and 600-21. The cultures which produce the virulent virus continue to do so indefinitely in the absence of the mutagen, but are not lysogenic for the virus. Most of the cells in such cultures are resistant to the virus and do not produce any, but there are a few mutant cells sensitive to the virus and the virus multiplies by infection of these sensitive mutants.     

The Structure of Mytilus Smooth Muscle and the Electrical Constants of the Resting Muscle

The individual muscle fibers of the anterior byssus retractor muscle (ABRM) of Mytilus edulis L. are uninucleate, 1.2–1.8 mm in length, 5 µm in diameter, and organized into bundles 100–200 µm in diameter, surrounded by connective tissue. Some bundles run the length of the whole muscle. Adjacent muscle cell membranes are interconnected by nexuses at frequent intervals. Specialized attachments exist between muscle fibers and connective tissue. Electrical constants of the resting muscle membrane were measured with intracellular recording electrodes and both extracellular and intracellular current-passing electrodes. With an intracellular current-passing electrode, the time constant τ, was 4.3 ± 1.5 ms. With current delivered via an extracellular electrode τ was 68.3 ± 15 ms. The space constant, λ, was 1.8 mm ± 0.4. The membrane input resistance, R_eff, ranged from 23 to 51 MΩ. The observations that values of τ depend on the method of passing current, and that the value of λ is large relative to fiber length and diameter are considered evidence that the individual muscle fibers are electrically interconnected within bundles in a three-dimensional network. Estimations are made of the membrane resistance, R_m, to compare the values to fast and slow striated muscle fibers and mammalian smooth muscles. The implications of this study in reinterpreting previous mechanical and electrical studies are discussed.     

Bacterial Cell Division Regulation: Lysogenization of Conditional Cell Division lon− Mutants of Escherichia coli by Bacteriophage Lambda

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.     

A probabilistic model of local sequence alignment that simplifies statistical significance estimation

Sequence database searches require accurate estimation of the statistical significance of scores. Optimal local sequence alignment scores follow Gumbel distributions, but determining an important parameter of the distribution (λ) requires time-consuming computational simulation. Moreover, optimal alignment scores are less powerful than probabilistic scores that integrate over alignment uncertainty (“Forward” scores), but the expected distribution of Forward scores remains unknown. Here, I conjecture that both expected score distributions have simple, predictable forms when full probabilistic modeling methods are used. For a probabilistic model of local sequence alignment, optimal alignment bit scores (“Viterbi” scores) are Gumbel-distributed with constant λ=log 2, and the high scoring tail of Forward scores is exponential with the same constant λ. Simulation studies support these conjectures over a wide range of profile/sequence comparisons, using 9,318 profile-hidden Markov models from the Pfam database. This enables efficient and accurate determination of expectation values (E-values) for both Viterbi and Forward scores for probabilistic local alignments.     

High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage

Genetic modifications of bacterial chromosomes are important for both fundamental and applied research. In this study, we developed an efficient, easy-to-use system for genetic modification of the Escherichia coli chromosome, a two-plasmid method involving lambda Red (λ-Red) recombination and I-SceI cleavage. An intermediate strain is generated by integration of a resistance marker gene(s) and I-SceI recognition sites in or near the target gene locus, using λ-Red PCR targeting. The intermediate strain is transformed with a donor plasmid carrying the target gene fragment with the desired modification flanked by I-SceI recognition sites, together with a bifunctional helper plasmid for λ-Red recombination and I-SceI endonuclease. I-SceI cleavage of the chromosome and the donor plasmid allows λ-Red recombination between chromosomal breaks and linear double-stranded DNA from the donor plasmid. Genetic modifications are introduced into the chromosome, and the placement of the I-SceI sites determines the nature of the recombination and the modification. This method was successfully used for cadA knockout, gdhA knock-in, seamless deletion of pepD, site-directed mutagenesis of the essential metK gene, and replacement of metK with the Rickettsia S-adenosylmethionine transporter gene. This effective method can be used with both essential and nonessential gene modifications and will benefit basic and applied genetic research.     

Loss of the Polarity Protein PAR3 Activates STAT3 Signaling via an Atypical Protein Kinase C (aPKC)/NF-κB/Interleukin-6 (IL-6) Axis in Mouse Mammary Cells

PAR3 suppresses tumor growth and metastasis in vivo and cell invasion through matrix in vitro. We propose that PAR3 organizes and limits multiple signaling pathways and that inappropriate activation of these pathways occurs without PAR3. Silencing Pard3 in conjunction with oncogenic activation promotes invasion and metastasis via constitutive STAT3 activity in mouse models, but the mechanism for this is unknown. We now show that loss of PAR3 triggers increased production of interleukin-6, which induces STAT3 signaling in an autocrine manner. Activation of atypical protein kinase C ι/λ (aPKCι/λ) mediates this effect by stimulating NF-κB signaling and IL-6 expression. Our results suggest that PAR3 restrains aPKCι/λ activity and thus prevents aPKCι/λ from activating an oncogenic signaling network.     

Genomic clones of a wild-type allele and a transposable element-induced mutant allele of the sucrose synthase gene of Zea mays L

In an attempt to isolate the transposable genetic element Ds from Zea mays L., we cloned DNA fragments hybridizing to a cDNA clone derived from the sucrose synthase gene in a λ vector (λ::Zm Sh). The fragments cloned from wild-type and from the Ds-induced mutant sh-m5933 (λ::Zm sh-m5933) share a segment 6 kb long while a contiguous segment of 15 kb of λ::Zm sh-m5933 (mutant-derived DNA) does not hybridize to the DNA segment cloned from the wild-type. Restriction maps are given, and the junction point between the two DNA segments in the mutant clone was determined. Hybridization of DNA fragments, present in the wild-type DNA of λ::Zm Sh, but not in the mutant clone, λ::Zm sh-m5933, to genomic DNA of sh-m5933 showed that no part of this DNA is deleted. It cannot be said whether the DNA found in the mutant, but not in the wild-type clone, has been brought there by Ds insertion or by another Ds-dependent DNA rearrangement. The mutant-derived DNA was hybridized to genomic DNA of various maize lines digested by several restriction endonucleases. Approximately 40 bands were detected. The mutant-derived DNA contains two pairs of inverted repeats several hundred nucleotide pairs long, one of which is located at the junction to wild-type-derived DNA.     

The Stochastic Theory of Cell Proliferation

A stochastic theory of cell kinetics has been developed based on a realistic model of cell proliferation. A characteristic transit time, _i, has been assigned to each of the four states (G₁, S, G₂, M) of the cell cycle. The actual transit time, t_i, for any cell is represented by a distribution around _i with a variance σ_i². Analytic and computer formulations have been used to describe the time development of such characteristics as age distribution, labeling experiments, and response to perturbations of the system by, for example, irradiation and temperature. The decay of synchrony is analyzed in detail and is shown to proceed as a damped wave. From the first few peaks of the synchrony decay one can obtain the distribution function for the cell cycle time. The later peaks decay exponentially with a characteristic decay constant, λ, which depends only on the average cell-cycle time, , and the associated variance. It is shown that the system, upon any sudden disturbance, approaches new “equilibrium” proliferation characteristics via damped periodic transients, the damping being characterized by λ. Thus, the response time of the system, /λ, is as basic a parameter of the system as the cell-cycle time.