Mutations in the right operator of bacteriophage lambda: physiological effects

The right operator in bacteriophage lambda vs326 has one-twentieth the in vitro binding affinity for repressor as λv⁺; for comparison λv3 has one-quarter the affinity of λv⁺. In vivo, both mutants constitutively express genes in the right operon. Both λv3 and λvs326 express gene O constitutively because they complement λimm434Oam^? in a λ lysogen, vs, more efficiently than v3. The v3 allele in cis (but not in trans) to vs326 gives significantly greater phage yields in a λ lysogen than λvs326 alone, cro gene function, measured by arrest of exonuclease synthesis, suggested the following series of increasing degree of conatitutivity: v3, vs326, v3 vs326. λv2 vs326 forms plaques on lysogens that carry λcI857, but λv2 v3 does not. These results indicate that vs326, like v3, is an operator constitutive mutation but stronger in its effects. These mutants exemplify a uniform correlation between relative weakness of repressor binding and degree of constitutive gene expression.     

An analysis of repressor overproduction by the lambda cIIIs mutant.

Efficient lysogenization of Escherichia coli K12 by bacteriophage λ requires the high level of synthesis of the phage repressor shortly after infection. This high level of synthesis of repressor requires the action of the λ eII and cIII proteins. Certain mutants of λ (λcIIIs) appear to have excess $\text{c}\text{II}\text{c}\text{III}$ activity and can lysogenize more efficiently than λ⁺. The basis for the enhanced lysogenization is that, while two or more infecting phage are necessary for λ⁺ to lysogenize, a single infecting λcIIIs particle is sufficient for lysogenization. Also, repressor levels in cells infected with λcIIIs are higher than in those infected with λ⁺. I report here that repressor overproduction by λcIIIs (1) is due to a much higher rate of repressor synthesis than that of λ⁺; (2) is most marked at low multiplicities of infection, possibly because λcIIIs produces repressor much more efficiently than λ⁺ as a singly infecting phage.     

The separation of phage promoter from bacterial lac promoter for beta-galactosidase expression in transducing phage lambda plac5.

The replication defective transducing phage λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 carries a portion of the $\text{E.}\text{coli}\text{lac}$ operon in the $\text{b}$2 region of the lambda phage. This $\text{lac}$ operon segment contains the $\text{lac}$ promoter, the $\text{lac}$ operator, and the β-galactosidase $\text{z}$ gene, but does not contain the $\text{lac}$ repressor $\text{i}$ gene. The $\text{z}$ gene can be expressed from both the inserted $\text{lac}$ promoter and the phage promoter. When $\text{E.}\text{coli}$ strain 594 ($\text{z}$^?, $\text{i}$⁺) or JC6256 (Δ$\text{lac}$) is infected by λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 in the absence of additional cyclic AMP, β-galactosidase synthesis is shown to be expressed from the phage promoter. When 594 (λ⁺) or JC6256 (λ⁺) is infected by λp$\text{lac}\text{5}\text{O}\text{29}\text{P}$3 in the presence of additional cyclic AMP and IPTG, β-galactosidase synthesis is shown to be expressed from the inserted $\text{lac}$ promoter.The ability to separate the phage promoter from the inserted $\text{lac}$ promoter for β-galactosidase expression will simplify the interpretation whenever λp$\text{lac}$5 is used.     

Binding of a simian virus 40 T antigen-related protein to DNA

Crystals of the periplasmic galactose-binding protein of Salmonella typhimurium have been subjected to X-ray analysis. The crystals grow as elongated rectangular prisms, with the symmetry of space group C2. Unit cell dimensions are $\text{a = 119·7}\text{A}\text{?}\text{, b = 37·2}\text{A}\text{?}\text{, c = 80·1}\text{A}\text{?}\text{, and}\text{β = 123·4}$. There is one protein molecule of molecular weight 33,000 per asymmetric unit.     

Crystallizations and preliminary X-ray studies of calotropins DI and DII

Crystals of calotropin DI (M_r 23,400), have been prepared by microdialysis against 5% (w/v) polyethylene glycol 20,000 in water, pH 7.0. They have orthorhombic space group P2₁2₁2₁ with cell parameters $\text{a = 57.5}\text{A}\text{?}\text{, b = 86.2}\text{A}\text{?}\text{, c = 40.3}\text{A}\text{?}$. Crystals of calotropin DII (M_r 24,000), prepared by the same technique against 5% (w/v) polyethylene glycol 20,000 in phosphate buffer of low ionic strength, pH 7.0, display monoclinic space group C2 with cell parameters $\text{a = 135.8}\text{A}\text{?}\text{, b = 32.0}\text{A}\text{?}\text{, c = 47.7}\text{A}\text{?}\text{, β = 103.80 °}$. In both cases, there is only one molecule in the asymmetric unit.     

Betaine-homocysteine methyltransferase in the fungus Aspergillus nidulans.

A betaine:homocysteine methyltransferase activity was demonstrated in the cell-free extracts from the fungus $\text{Aspergillus}\text{nidulans}$. Among methionine-requiring mutants which do not grow on homocysteine one class responds to betaine indicating that this compound can serve as a methyl donor in methionine synthesis $\text{in vivo}$. Mutants of the second class which grow only on methionine were shown to have betaine: homocysteine — and methyltetrahydrofolate: homocysteine methyltransferases simultaneously impaired.     

The decay of genetic variability in geographically structured populations. II

The ultimate rate of approach to equilibrium in the infinite stepping-stone model is calculated. The analysis is restricted to a single locus in the absence of selection, and every mutant is assumed to be new to the population. Let f(t, x) be the probability that two homologous genes separated by the vector x in generation t are the same allele. It is supposed that $\text{f(}\text{0, x}\text{) = O(x}{}^{\mathrm{?2?\eta }}\text{), η > 0,}\text{as}\text{x ≡ ¦}\text{x}\text{¦ → ∞}$. In the absence of mutation, f(t, x) tends to unity at the rate $\text{t}{}^{\mathrm{<mtext>?1</mtext><mtext>2</mtext>}}$ in one dimension and (ln t)^?1 in two dimensions. Thus, the loss of genetic variability in two dimensions is so slow that evolutionary forces not considered in this model would supervene long before a two-dimensional natural population became completely homogeneous. If the mutation rate, u, is not zero f(t, x) asymptotically approaches equilibrium at the rate $\text{(1 ? u)}{}^{\mathrm{2t}}\text{t}{}^{\mathrm{<mtext>?3</mtext><mtext>2</mtext>}}$ in one dimension and (1 ? u)^2tt^?1(lnt)^?2 in two dimensions. Integral formulas are presented for the spatial dependence of the deviation of f(t, x) from its stationary value as t → ∞, and for large separations this dependence is shown to be (const + x) in one dimension and (const + ln x) in two dimensions. All the results are the same for the Malécot model of a continuously distributed population provided the number of individuals per colony is replaced by the population density. The relatively slow algebraic and logarithmic rates of convergence for the infinite habitat contrast sharply with the exponential one for a finite habitat.     

Non-autonomous logistic equations as models of populations in a deteriorating environment

The non-autonomous logistic equation

$\text{d}\text{x(t)}\text{d}\text{t}\text{= r(t)x(t)[1 ?}\text{x(t)}\text{K(t)}\text{]}$

is studied under conditions that include an environment which is completely deteriorating. In this setting, when the population's growth rate, r, is large on the average, solutions track the environment with a consequent extinction of the population. However, when both r and rK^?1 are small in the sense that they are in L¹[0,∞) then an asymptotic equivalence, where all solutions tend to positive limits as t approaches infinity, results and the population is persistent, independent of initial density. The asymptotic equivalence produces an unreasonable overshoot of carrying capacity which leads to concern about employing the logistic equation in the above form as a population model when growth rates are close to zero.A re-interpretation of the parameters of the logistic equation leads to the alternative logistic formulation

$\text{d}\text{x(t)}\text{d}\text{t}\text{= x(t)[r(t) ?}\text{c}\text{B(t)}\text{x(t)], (c > 0)}$

. A biological interpretation of the parameters is presented and this equation is compared with the classical logistic model in the case where the parameters are constant. If the alternative logistic model is applied in a situation with time-varying parameters, then a deteriorating environment always leads to extinction of the population regardless of the behavior of r. Similarly, a growth rate which is small on the average results in extinction regardless of the behavior of B. Furthermore, r and B have limiting values as t approaches infinity then so does x and the terminal value of x is equal to the terminal value of the carrying capacity of the population. In general, the alternative formulation seems to be the more reasonable model in situations where perturbations lead to severe decreases in environmental quality and growth rates.     

The accumulation of deleterious genes in a population—Muller's Ratchet

A quantitative study of the operation of Muller's Ratchet for the accumulation of deleterious genes in an asexually reproducing population is made. For a population of size N, in which deleterious mutations occur at rate λ/genome/ generation, and the relative fitness of an individual with k mutants is (1 ? s)^k, the most important parameter is $\text{n}{}_0\text{= Ne}{}^{\mathrm{?\theta }}\text{,}\text{where}\text{θ =}\text{λ}\text{s}$. If n₀ is large (?25), deleterious mutations will accumulate very slowly, and independently of each other; if n₀ is small (<1), the rate of accumulation of deleterious mutations will be greater than a natural population could plausibly bear; an estimate of the speed of the Ratchet for intermediate values of n₀ is made. It is pointed out that the frequency distribution for the numbers of individuals carrying k mutants will retain its shape, but will move bodily to the right at the same average speed as the Ratchet. When favourable mutations also occur, the frequency distributions can move right of left; an estimate of the probability that any particular step is right or left is made, and it is shown that, for a given net rate of arrisal of deleterious mutations, the greater the rate of beneficial mutation, the greater the chance that beneficial mutations will accumulate.     

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.     

Binding equations for interacting systems comprising multivalent acceptor and bivalent ligand: application to antigen-antibody systems

Consideration is given to the reversible interaction of a bivalent ligand, B, with a multivalent acceptor, A (possessing f reactive sites) which leads to the formation of a series of complexes, A_iB_j, comprising networks of alternating acceptor and ligand molecules. A binding equation is derived on the basis of a site association constant, k, defined in terms of reacted site probability functions. This equation, which relates the binding function, r (the moles of ligand bound per mole of acceptor) to the concentration of unbound ligand, m_b, is used to show that plots of r vs. 2km_B constructed with fixed but different values of $\text{k}\text{m}{}_{\mathrm{A}}$ intersect at the point ($\text{m}{}_{\mathrm{B}}\text{=}\text{1}\text{2k}\text{, r =}\text{f}\text{2}$) where the extent of reaction and the concentrations of those complexes for which $\text{j}\text{i}\text{=}\text{f}\text{2}$ attain maximal values. Corresponding Scatchard plots are shown by numerical example to be non-linear, their second derivative being positive for all r. It follows that such deviations from linearity cannot be taken alone as evidence for site heterogeneity in cross-linking systems. The binding equation obtained directly is shown to be identical with that obtained with f = 2 by summation procedures involving the general expression for concentrations of complexes, m_AiBj, formulated in terms of appropriate statistical factors. In this way, previous findings on precipitation and gel formation in cross-linking systems are correlated with the present development of binding theory.     

A dynamical behaviour of active regions in randomly connected neural networks

The field of the randomly connected neural network is approximately formulated by Griffith's equation, regarding the network as being continuous. An integral representation of Griffith's equation is derived. If a relative refractory period can be ignored, it is $\text{X(x,t)}\text{=1}\text{∫}\text{o}\text{∞}\text{ds}\text{∫}\text{?vs}\text{vs}\text{dn}\text{kv}\text{2t}\text{e}{}^{\mathrm{?avs}}\text{X(x?n, t?s) ? θ}$ where X(x, t) corresponds to the firing rate and θ means the threshold of the neural firing, τ the absolute refractory period and v the velocity for the spike potential travelling down the axon. The above equation is formally analogous to Caianiello's equation, but the former describes the more macroscopic behaviour of the neural network than the latter. With the aid of computer simulation, appropriate solutions are successfully obtained.In regions where X = 1, neurones are firing at a high constant rate of $\text{1}\text{τ}$ (active regions). In regions where X = 0, there is no firing of neurones (resting regions). In the neural net for which $\text{0 <}\text{a}{}^2\text{τθ}\text{k}\text{< 1}$, the net is generally a mixture of the active regions and of the resting regions. In the case that a large active region is in contact with a large resting region, the propagation velocity of boundary between the two regions tends to the velocity u given by $\text{u = (1 ?}\text{2a}{}^2\text{τθ}\text{k}\text{)v}$. This expression of velocity u was deduced from the fact that there exists a solution of the type X(x, t) = 1 (ut ? x) for equation (A). In the case of $\text{0 <}\text{a}{}^2\text{τθ}\text{k}\text{< 0 · 5}$, the active region grows and in the case of $\text{0 · 5 <}\text{a}{}^2\text{τθ}\text{k}\text{< 1}$, the resting region grows. A fatigue effect is introduced, for which it is hard for neurones to maintain firing states. In this case an active region of definite width L propagates with constant velocity u′. The dependence of L and u′ on characteristics of neural network and on the fatigue effect is investigated.     

Plasmid vectors for high-efficiency expression controlled by the PL promoter of coliphage lambda

A family of plasmid cloning vectors has been constructed that make use of the leftward promoter ($\text{P}{}_{\mathrm{<mtext>L</mtext>}}$) of phage λ to provide for efficient expression of cloned genes in Escherichia coli. The promoter activity of $\text{P}{}_{\mathrm{<mtext>L</mtext>}}$ is fully repressed at low temperature by a thermolabile repressor product of the $\text{λc}\text{I}\text{1857}$ gene, and can be activated by heat induction. Examples are given (β-lactamse, tryptophan synthetase A) where, under optimal conditions, between 30 and 40% of the total protein synthesis is directed by the cloned gene under $\text{P}{}_{\mathrm{<mtext>L</mtext>}}$ control.     

Mutagenicity of the carcinogen 7-bromomethylbenz (a)-anthracene: a quantitative study in repair-deficient strains of Escherichia coli

The mutagenicity of 7-bromomethylbenz[a]anthracene, a reactive arylalkylating carcinogen, was investigated in several strains of E. coli WP2, using reversion from tryptophan auxotrophy (ochre trpE locus) as a measure of induced mutation.WP2, the wild-type with respect to DNA repair, was more resistant to the cytotoxic effects of 7-bromomethylbenz[a]anthracene than WP2uvrA, WP2exrA, or WP2uvrAexrA, the D₃₇ doses of carcinogen being 22, 5, 8, and 1 μg/ml respectively. Mutagenesis in WP2 was observed only at doses in excess of the D_Q, whereas in WP2uvrA mutation was linearly related to dose throughout the range studies. No mutation was detectable in WP2exrA or WP2uvrAexrA even at doses which resulted in 95% and 99.9% lethality respectively. It was concluded that an intact Exr function was an absolute requirement for the induction of mutation by 7-bromomethylbenz[a]anthracene and that excision-repair was very efficient in removing premutational lesios.The use of $\text{[}{}^3\text{H}\text{]7-}\text{bromomethylbenz}\text{[a]}\text{anthracene}$ at high specific radioactivity enabled the quantitation of mutation as a function of the extent of reaction of the mutagen with cellular macromolecules. Extent of reaction with DNA, RNA and protein was linearly related to dose, binding to DNA being 3 times that to RNA and 20 times that to protein. There was a linear relationship between binding and mutation in WP2uvrA and the effective target size for Exr-mediated mutation in this system was of the order of 0.04 nucleotides. Having established that the n umber of 7-bromomethylbenz[a]anthracene-induced mutants increased linearly with successive cell generations and, by use of T4 ochre427, that about 30% of the mutants scored were true revertants, it was estimated that the Exr pathway incorporates productive errors into the bacterial genome with a frequency of the order of 2.10^?3.     

Preliminary X-ray data for the ribose binding protein from Salmonella typhimurium

Crystals of the periplasmic ribose binding protein of Salmonella typhimurium have been subjected to X-ray analysis. The crystals grow as rectangular parallelopipeds with the symmetry of space group P2₁. Unit cell dimensions are $\text{a = 64·4}\text{A}\text{?}\text{, b = 60·6}\text{A}\text{?}\text{, c = 62·8}\text{A}\text{?}$, and β = 91·25 °. There are two molecules of molecular weight 29,000 per asymmetric unit.     

Chemiluminescence in the reaction of cytochrome c with hydrogen peroxide

(1) Aqueous solutions of 1–10 μM ferricytochrome c treated with 100 μM–100 mM H₂O₂ at pH 8.0 emit chemiluminescence with quantum yield $\text{Ф ? 10}{}^{\mathrm{?9}}$ and absolute maximum intensity $\text{I}{}_{\mathrm{<mtext>max</mtext>}}\text{? 10}{}^5\text{hv/}\text{s}$ per cm³ (λ = 440), and exhibit exponential decay with a rate constant of 0.15 s^?1. (2) The emission spectrum of the chemiluminescence covers the range 380–620 nm with the maximum at 460 ± 10 nm. (3) Neither cytochrome c nor haemin fluoresce in the spectral region of the chemiluminescence. In the reaction course with H₂O₂, a weak fluorescence in the region 400–620 nm with λ_max = 465–510 nm (λ_exc 315–430 nm) gradually arises. This originates from tryptophan oxidation products of the formylkynurenine type or from imidazole derivatives, respectively. (4) Frozen solutions (77 K) of cytochrome c exhibit phosphorescence typical of tryptophan (λ_exc = 280 nm, λ_em = 450 nm). During the peroxidation, an additional phosphorescence gradually appears in the range 480–620 nm with λ_max = 530 nm (λ_exc = 340 nm). This originates from oxidative degradation products of tryptophan. (5) There are no red bands in the chemiluminescence spectra of cytochrome c or haemin. This result suggests that singlet molecular oxygen $\text{O}{}_2\text{(}{}^1\text{Δ}{}_{\mathrm{<mtext>g</mtext>}}\text{)}$ is not involved in either peroxidation or chemiluminescence. (6) The haem Fe³⁺ group and H₂O₂ appear to be crucial for the chemiluminescence. It is suggested that the generation of electronically excited, light-emitting states is coupled to the production of conformational out-of-equilibrium states of peroxy-Fe-protoporphyrin IX compounds.     

Glycoprotein characteristics of the sodium channel saxitoxin-binding component from mammalian sarcolemma

The saxitoxin-binding component of the excitable membrane sodium channel exhibits glycoprotein characteristics as evidenced by its specific interaction with various agarose-immobilized lectins. The detergent-solubilized saxitoxin-binding component interacts quantitatively with immobilized wheat germ agglutinin and concanavalin A and fractionally with immobilized Lens culinaris hemagglutinin and Ricinus communis agglutinin. These lectins preferentially bind $\text{N-}\text{acetylglucosamine}$ and sialic acid (wheat germ agglutinin), mannose (concanavalin A and Lens cunilaris and galactose (Ricinus communis). Removal of terminal sialic acid residues by neuraminidase markedly decreases binding to immobilized wheat germ agglutinin but uncovers sites capable of interacting with lectins specific for galactose and $\text{N-}\text{acetylgalactosamine}$. $\text{β-N-}\text{acetylglucosaminidase}$, an exoglycosidase has no effect on the binding of the channel protein to wheat germ agglutinin. Similarly, phospholipase C has no effect on binding of the solubilized toxin binding component to this lectin. Neither wheat germ agglutinin nor concanavalin A free in solution alters the number of toxin binding sites or their affinity for toxin. The sodium channel saxitoxin-binding component appears to be a glycoprotein containing terminal sialic acid residues and internal mannose, galactose, $\text{N-}\text{acetylglucosamine}$, and $\text{N-}\text{acetylgalactosamine}$ residues. The toxin binding site is spatially separated from the binding sites for the lectins studied. The effect of these sugar moieties must be considered when evaluating the biophysical parameters of the sodium channel.     

Crystallographic analysis of the phytoagglutinin from Abrus precatorius by X-ray diffraction and electron microscopy

The lectin from the seeds of Abrus precatorius has been crystallized and the crystals subjected to study by X-ray diffraction and electron microscopy. Three closely related crystal forms were obtained, of orthorhombic space group P2₁2₁2₁ with $\text{a = 138}\text{A}\text{?}$, $\text{b = 142}\text{A}\text{?}$, and $\text{c = 173}\text{A}\text{?}$, of tetragonal space group P4₁2₁2 with $\text{a = b = 136}\text{A}\text{?}$, $\text{c = 176}\text{A}\text{?}$, and a twinned intermediate of the first two. From electron microscopy and two-dimensional spatial filtering of electron micrographs of the crystals, the molecule appears to consist of four similar domains grouped in a roughly planar diamond-shaped arrangement having a local intramolecular dyad axis. The average diameter of the Abrus lectin molecule is 50 to 60 Å and the individual domains appear to have a diameter of about 25 Å.     

Interaction of manganese with fragments, complementary fragment recombinations, and whole molecules of yeast phenylalanine specific transfer RNA

Binding of Mn²⁺ to the whole molecule, fragments and complementary fragment recombinations of yeast tRNA^Phe, and to synthetic polynucleotides was studied by equilibrium dialysis. The comparison of the binding patterns of the fragments, fragment recombinations and synthetic polynucleotides with that of intact tRNA^Phe permits reasonable conclusions concerning the nature and location of the various classes of sites on tRNA^Phe. Binding of Mn²⁺ to intact tRNA^Phe consists of a co-operative and a non-co-operative phase. There are about 17 “strong” sites and several “weak” ones. Five of the 17 strong sites are associated with the co-operative phase. This phase is completely lacking in the binding of Mn²⁺ to tRNA^Phe fragments (5′-$\text{1}\text{2}$, 3′-$\text{1}\text{2}$, 5′-$\text{3}\text{5}$, 3′-$\text{2}\text{5}$), poly-(A):poly(U) and poly(I):poly(C) helices, and single stranded poly(A) and poly(U). This argues that the co-operative sites arise from the tRNA tertiary structure. This conclusion is further strengthened by the observation that cooperativity is present in a tRNA^Phe molecule which has been split in the anticodon loop, but it is absent in one which has been split in the extra loop. It is in the vicinity of the latter loop, but not the former, that tertiary interactions are seen in the crystal structure. The remaining 12 strong sites are “independent” and appear to be associated with cloverleaf helical sections.