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The lysogenic state of phage λ is maintained by the CI repressor. CI binds to three operators each in the right operator ( O R ) and left operator ( O L ) regions, which lie 2.4 kb apart. At moderate CI levels, the predominant binding pattern is two dimers of CI bound cooperatively at each regulatory region. The resulting tetramers can then interact, forming an octamer and a loop of the intervening DNA. CI is expressed from the P RM promoter, which lies in the O R region and is subjected to multiple regulatory controls. Of these, the most recently discovered is stimulation by loop formation. In this work, we have investigated the mechanism by which looping stimulates P RM . We find that two cis -acting sites lying in the O L region are involved. One site, an UP element, is required for stimulation. Based on the behavior of other promoters with UP elements located upstream of the −35 region, we suggest that a subunit of RNA polymerase (RNAP) bound at P RM binds to the UP element located in the O L region. In addition, adjacent to the UP element lies a binding site for integration host factor (IHF); this site plays a less critical role but is required for stimulation of the weak prm240 allele. A loop with CI at the O L 2 and O L 3 operators does not stimulate P RM , while one with CI only at O L 2 provides some stimulation. We discuss possible mechanisms for stimulation.

Nanoscale engineering of band profile and potential profile provide effective tools for the management of photoelectron processes in quantum dot (QD) photovoltaic devices. We investigate the QD devices with various 1-μm InAs /GaAs QD media placed in a 3-μm base GaAs p-n junction. We found that n-charging of quantum dots (QDs) create potential barriers around QDs. QD growth between ultrathin AlGaAs layers leads to the formation of AlGaAs “fence” barriers, and reduces the wetting layers (WLs). The barriers around QDs and reduction of the wetting layer substantially suppress recombination processes via QDs. The n-doping of interdot space in QD media enhances electron extraction from QDs. All of our QD devices show short-circuit current, J SC , higher than that of the reference cell, but smaller open-circuit voltage, V OC .. In the QD devices, the short circuit currents increase by ~0.1 mA/cm 2 per dot layer. J SC reaches 28.4 mA/cm 2 in the device with QD media that combines dot charging, fence barriers, and WL reduction.

The CI repressors of lambdoid phages bind cooperatively to adjacent binding sites. Although these binding sites are found at complex operators containing three binding sites, cooperative binding occurs only between dimers bound at two of the sites, a mode of binding termed pairwise cooperativity. At the level of regulation, pairwise cooperativity allows the proper operation of the genetic switch. At the mechanistic level, it has been proposed to result from steric distortion of the complex, such that a protein dimer bound to a central site cannot contact both flanking dimers because it “leans” towards one of the two sites. We have tested this model using the CI repressor of phage HK022. Previous work suggested that reducing the spacing between adjacent operators might allow cooperative interaction among three dimers, a mode of cooperativity we term extended. Using a set of synthetic templates, we have shown that reducing the spacing allows extended cooperativity among three binding sites. Accordingly, the mode of cooperativity changes qualitatively in response to changes in the inter-site spacing. That is, the change in spacing has major functional consequences for the properties of the complex. We suggest that such changes can play important roles in the evolution of gene regulatory circuitry and of other processes involving nucleoprotein complexes.

Cooperative protein-DNA interactions play critical roles in gene regulation in all organisms. Among the best-studied cooperative interactions is that of phage λ repressor, which binds cooperatively to two adjacent operators. Similar cooperative interactions are also shown by several other lambdoid phage repressors, including HK022 CI repressor, which we study here. This protein has a much higher degree of cooperativity than seen with λ repressor, and previous evidence has suggested that cooperativity may play roles in HK022 gene regulation that have no parallel in λ. We have isolated several cooperativity or Coop− mutations in HK022 cI. These mutant proteins were partially defective in vivo for binding to two adjacent operators, but normal or nearly so for binding to a single operator. Two mutations showed mutual suppression, in that the double mutation had wild-type behavior. Analysis of several purified mutant proteins showed that they were also defective for cooperative binding in vitro. Unexpectedly, the mutant proteins showed an altered pattern of in vitro binding to DNA at non-operator sites. Several of them also increased the rate of specific repressor cleavage. We propose a conformational model in which the various functions of the wild-type protein are carried out by differing conformations; these conformations are normally in balance, and the mutations perturb this balance.

Cooperative binding of specific DNA-binding proteins plays crucial roles in gene regulatory circuitry, and is a model system for interactions between proteins bound to DNA. We have studied coliphage HK022 repressor, which binds to two adjacent operators with a cooperativity parameter of ≈ 2000. We examined the effect of changing the spacing between these two operators on cooperativity and on the conformation of the complex. Maximum cooperativity was seen with the wild-type spacing; considerable cooperativity was retained for most spacing variants, but was abolished when the operators lay on opposite faces of the DNA helix. Most spacing variants conferred changes in the conformation of the DNA-protein complex. Our data indicate that the pairwise cooperativity observed with the wild-type spacing results from a conformation that prevents protein-protein contacts with flanking bound dimers. We conclude that protein-DNA complexes involving the same specific binding sites and the same protein molecules can adopt many different conformations, depending on the spacing between the binding sites. This conclusion may be broadly applicable to protein-DNA interactions in other systems.

This report describes the highlights and summary of the year 2, quarter four (Y2Q4) progress for the 6.2 project Improved Sensitivity Low-Cost Uncooled IR Detector Focal-Plane Arrays. This work occurred during the January 2010 May 2010 time period. The program goals for Y2Q4 corresponded to the continued expansion of our barium titanium oxide (BaTiO3) nanomaterial synthesis capabilities and deposition methods; laser-induced pyroelectric phase conversion studies and nanoscale characterization of the pyroelectric activation process; and fabrication of infrared (IR) test heterostructures for pyroelectrical analysis, along with the development of a technique for generating the requisite IR absorbing layer.

Diminished performance due to poor chemical and electrical surface stability of InAs/GaSb SL photodetectors continues to be a major hurdle to the realization of the theoretically predicted high performance of this material system. Improved epitaxial growth conditions have yielded improvements in material quality over the past several years. However, surface instability resulting in electrical shunt pathways across the junction, and diminished device performance over time is still a major limiting factor for application of InAs/GaSb SL in long-wavelength infrared detectors. This study focuses on a two-step approach towards the successful surface passivation of long-wavelength InAs/GaSb superlattice structures. Two distinct sulfide chemical surface treatments were applied to inhibit the formation of native surface oxides and satisfy dangling bonds. This was followed by the application of a robust SU8-2 dielectric treatment on the mesa sidewalls to inhibit sulfide layer degradation and oxidation of the surface over time. A variable area diode analysis (VADA) technique employing diodes of variable diameter (40-400um) enabled the investigation of surface resistivity as a result of different passivation treatments. Temperature dependent studies of the dark current were used to understand the dominating current mechanisms.

We report a 50% increase in power efficiency for our novel InAs/GaAs quantum dot with built-in charge (Q-BIC) solar cells. We found that n-doping the inter-dot space of a quantum dot solar cell (QDoSC) increases the short circuit current density from 15.07 mA/cm2 in undoped QDoSC to 24.30 mA/cm2 in the device doped to provide approximately six electrons per dot. To identify the physical mechanisms that provide this significant improvement, we investigate the photovoltaic response and its spectral characteristics in GaAs reference cell, undoped, n-doped, and p-doped QDoSCs. We found that the photovoltaic efficiency of the undoped QDoSC is almost the same as that of the reference cell. The efficiency monotonically improves with increasing n-doping, while the p-doping deteriorates the photovoltaic conversion. Studies of the photoluminescence of p- and n-doping show that the photoelectron capture into QDs is substantially faster than the hole capture, which leads to an accumulation of electrons in QDs. The built-in-dot electron charge enhances electron inter-subband QD transitions, suppresses the fast electron capture processes, and together with charged donors, forms the potential profile which precludes degradation of the open circuit voltage. All of these factors lead to the enhanced harvesting of IR energy and to a radical improvement of the QDoSC efficiency. Even higher efficiencies are anticipated for higher n-doping levels.

Pyroelectric infrared (IR) detectors based on perovskite oxides are of interest in part because of their lack of need for cooling, which makes them relatively more affordable and operationally simpler than cooled photon detector systems. We are investigating two methods for low-cost growth of perovskite oxide thin films, namely, a bio-inspired, low-temperature synthesis method and a modified industry-standard metalorganic solution deposition (MOSD) method. Subsequent to film synthesis, we utilize direct-write laser phase conversion and micro-electro-mechanical systems (MEMS) fabrication for development of an uncooled IR focal plane array (FPA). Film growth, crystallization and MEMS processes are compatible with monolithic integration of the detector pixels directly onto Si readout integrated circuits (ROICs).

We report the results of a study of the temperature-dependent photoluminescence (PL) and leakage current characteristics of a set of type II idium arsenide (InAs)-gallium antimonide (GaSb) superlattice (SL) photodiode structures. We find that the PL efficiency of high-quality structures is determined by Shockley-Read and trap-assisted tunneling nonradiative recombination processes. Our results suggest a possible correlation between trap-assisted tunneling in some SL structures and an anomalous decrease in the PL efficiency with increasing temperature over the range 40-78 K, and provide insight into the effect of defects and SL homogeneity on the PL and transport characteristics of the photodiodes.

The LexA repressor of Escherichia coli modulates the expression of the SOS regulon. In the presence of DNA damaging agents in vivo, the 202-amino acid LexA repressor is inactivated by specific RecA-mediated cleavage of the Ala-84/Gly-85 peptide bond. In vitro. LexA cleavage requires activated RecA at neutral pH, and proceeds spontaneously at high pH in an intramolecular reaction termed autodigestion. A model has been proposed for the mechanism of autodigestion in which serine 119 serves as the reactive nucleophile that attacks the Ala-84/Gly-85 peptide bond in a manner analogous to a serine protease, while uncharged lysine 156 activates the serine 119 hydroxyl group. In this work, we have tested this model by examining the effect of the serine protease inhibitor diisopropyl fluorophosphate (DFP) on autodigestion. We found that DFP inhibited autodigestion and that serine 119 was the only serine residue to react with DFP. We also examined [3H]DFP incorporation by a number of cleavage-impaired LexA mutant proteins and found that mutations in the proposed active site, but not in the cleavage site, significantly reduced the rate of [3H]DFP incorporation. Finally, we showed that the purified carboxyl-terminal domain, which contains the proposed catalytic residues, incorporated [3H]DFP at a rate indistinguishable from the intact protein. These data further support our current model for the mechanism of autodigestion and the organization of LexA.

A critical step in the SOS response of Escherichia coli is the specific proteolytic cleavage of the LexA repressor. This reaction is catalyzed by an activated form of RecA, acting as a co-protease to stimulate the self-cleavage activity of LexA. This process has been reexamined in light of evidence that LexA is dimeric at physiological concentrations. We found that RecA-dependent cleavage was robust under conditions in which LexA is largely dimeric and conclude that LexA dimers are cleavable. We also found that LexA dimers dissociate slowly. Furthermore, our evidence suggests that interactions between the two subunits of a LexA dimer can influence the rate of cleavage. Finally, our evidence suggests that RecA stimulates the transition of LexA from its noncleavable to its cleavable conformation and therefore operates, at least in part, by an allosteric mechanism.

Under relatively high pump intensities, photoluminescence intensities across the miniband were greatly reduced, which was attributed to the existence of periodic electric fields caused by spatially-separated photogenerated electrons and holes in short-period type-II InAs/GaSb superlattices.

Effects of concentrated solar radiation on photovoltaic performance are investigated in well-developed GaAs quantum dot (QD) solar cells with 1-Sun efficiencies of 18%–19%. In these devices, the conversion processes are enhanced by nanoscale potential barriers and/or AlGaAs atomically thin barriers around QDs, which prevent photoelectron capture to QDs. Under concentrated radiation, the short circuit current increases proportionally to the concentration and the open circuit voltage shows the logarithmic increase. In the range up to hundred Suns, the contributions of QDs to the photocurrent are proportional to the light concentration. The ideality factors of 1.1–1.3 found from the VOC-Sun characteristics demonstrate effective suppression of recombination processes in barrier-separated QDs. The conversion efficiency shows the wide maximum in the range of 40–90 Suns and reaches 21.6%. Detailed analysis of I-V-Sun characteristics shows that at low intensities, the series resistance decreases inversely proport...

We are investigating the structural, electrical, and infrared optical properties of a new material system comprising undoped self-assembled quantum dots having a type II band alignment with the matrix material in which they are embedded. This material system is fundamentally different from conventional type I quantum dots that must be doped and that rely on intersubband transitions for infrared photoresponse. Type II quantum dots would operate in photovoltaic mode with infrared photoresponse arising from electron-hole pair production involving 3-dimensionally-confined states in the dots and quantum well states in the matrix material. This paper emphasizes the structural characterization of molecular beam epitaxy (MBE)-grown InSb quantum dots grown in an In0.53Ga0.47As matrix lattice matched to an InP substrate.

The Type II broken band-gap alignment in semiconductor structures wherein the conduction band minimum is in one semiconductor (e.g., InAs) and the valence band maximum is in another (e.g., GaInSb) offers certain unique advantages which can be utilized to realize band-gap engineered novel quantum electro-optic devices such as lasers and detectors. The advantages of the type II structures include reduced Auger recombination, extending the effective band-gap energy of materials wherein type I band-gap alignment would give rise to difficulties such as miscibility gap. In this paper we describe the work carried out at the Army Research Laboratory on type II semiconductor quantum electro-optic devices such as IR lasers and detectors operating in the 3-12 μm range. Specifically we will cover the progress made in GaSb based type II strained layer superlattice IR detectors and Interband Cascade IR Lasers. We will also present our recent work in self-assembled quantum dots which have type II band-gap alignment with the matrix material in which the dots are embedded.

We demonstrate that longitudinal-optical phonons in type-II InAs/GaSb quantum wells can be used to up-convert photoluminescence. Such a phenomenon could be used to achieve laser-induced cooling of lattices.

We have studied the coupling between the InAs quantum dots and coupled quantum wells strained by the InAs quantum dots (i.e. coupled-quantum-well dots) by measuring photoluminescence spectra. By comparing the photoluminescence spectra of the coupled-quantum-well dots in which either the narrow well or wide well is grown next to the InAs quantum dots, we have evidenced the spatially inhomogeneous strains applied by the InAs quantum dots on the coupled quantum wells. Moreover, we have also investigated the three coupling regimes, i.e. when the dot level is higher than, lower than, and aligned to the lowest quantum-well transition. Based on our measurements, we conclude that the optical properties for the coupled-quantum-well dots have been significantly improved over the quantum dots.

We have demonstrated that type-II InAs/GaSb superlattices exhibit anomalously strong band-filling effects. By varying the width of the barrier or well, we have investigated the effects from 2-D quantum wells to quasi-3-D superlattices.

We report on the fabrication of quantum grid infrared photodetector (QGIP) arrays and demonstrate their feasibility for use as multi-channel long wavelength infrared spectrometers. The quantum well infrared photodetector (QWIP) material structure was designed to exhibit broadband absorption in the wavelength range of 7 μm to 16 μm. By fabricating QGIP devices with this QWIP material, scattering of light at an individual wavelength of interest within the material absorption range can create narrow band detection in each device. Arrays of QGIP devices with varying geometry, each tailored to respond to a discrete wavelength were fabricated. Details of the epi-growth, processing steps taken to fabricate required device features for narrow band absorption of the QGIP devices, and characterization methods will be discussed.

We have studied coupling between InGaAs/GaAs coupled quantum wells and self-assembled InAs quantum dots by measuring the linewidths, peak energies and peak intensities of photoluminescence peaks for different pump powers and lateral locations

Quantum dots (QDs) provide photovoltaic conversion of below-bandgap photons due to multistep electron transitions. QDs also increase conversion efficiency of the above-bandgap photons due to extraction of electrons from QDs via Coulomb interaction with hot electrons excited by high-energy photons. Nanoscale potential profile (potential barriers) and nanoscale band engineering (AlGaAs atomically thin barriers) allow for suppression of photoelectron capture to QDs. To study these kinetic effects and to distinguish them from the absorption enhancement due to light scattering on QDs, we investigate long, 3-μm base GaAs devices with various InAs QD media with 20 and 40 QD layers. Quantum efficiency measurements show that, at least at low doping, the multistep processes in QD media are strongly affected by the wetting layer (WL). The QD media with WLs provide substantial conversion of below-bandgap photons and for devices with 40 QD layers the short circuit current reaches 29.2 mA/cm2. The QD media with band-engineered AlGaAs barriers and reduced wetting layers (RWL) enhance conversion of high-energy photons and decrease the relaxation (thermal) losses.

This paper provides the design details for a new two-color quantum-well infrared photodetector for use in advanced thermal imaging. The single pixel experimental data used in the evolution of this design is discussed in significant detail. Using the knowledge gained from extensive measurements comparing miniband transport with bound-to-quasi-bound QWIPs and also double and triple coupled-well QWIPs with the more conventional single-well QWIPs, a two-color design is presented. The detector design provides for a lattice-matched growth and superior response in the MWIR spectral region, while keeping the dark currents from the LWIR portion of the detector to a minimum. The architecture will allow for the MWIR and LWIR stacks to be biased separately and also for the signals to be read independently.

We report on results of laboratory and field tests of dual- band MWIR/LWIR focal plane arrays (FPAs) produced under the Army Research Laboratory's Multidomain Smart Sensor Federated Laboratory program. The FPAs were made by DRS Infrared Technologies using the HgCdTe material system and by BAE Systems using QWIP technology. The HgCdTe array used the DRS HDVIPTM process to bond two single-color detector structures to a 640 X 480-pixel single-color read-out integrated circuit (ROIC) to produce a dual-band 320 X 240 pixel array. The MWIR and LWIR pixels are co-located and have a high fill factor. The images from each band may be read out either sequentially (alternating frames) or simultaneously. The alternating frame approach must be used to produce optimal imagery in both bands under normal background conditions. The QWIP FPA was produced using MBE-grown III-V materials. The LWIR section consisted of GaAs quantum wells and AlGaAs barriers and the MWIR section used InGaAs quantum wells with AlGaAs barriers. The detector arrays were processed with three ohmic contacts for each pixel allowing for independent bias control over both the MWIR and LWIR sections. The arrays were indium bump-bonded to an ROIC (specifically designed for two color operation) which puts out the imagery from both bands simultaneously. The ROIC has variable gain and windowing capabilities. Both FPAs were tested under similar ambient conditions with similar optical components. The FPAs were subjected to a standard series of laboratory performance tests. The relative advantages and disadvantages of the two material systems for producing medium-format dual-band FPAs are discussed.

We report the result of intersubband absorption measurement on a series of doped quantum-well samples and the determination of both the nonparabolicity parameters and the conduction band/valence band offset ratio of the GaAs/(AlGa)As material system. Absorption spectra were obtained in an FTIR spectrometer for samples with nominal quantum-well widths of 4.0, 4.5, 5.0, 7.5, 10.0, and 15.0 nm and for a series of miniband-transport quantum-well IR photodetectors. X-ray diffraction was used to determine quantum well and barrier thicknesses and the Al mole fraction x of the (Al,Ga)As barrier layers. Absorption measurements were made at temperatures of 295 K and 77 K. An empirical two-band model with an energy-dependent effective mass was used to calculate energy levels, transition energies, and spectral lineshapes for the quantum-well structures. Parameter values for nonparabolicity and band offset ratio were determined by comparison of calculated transition energies to measured spectral data.

Infrared sensor technology is critical to many commercial and military defense applications. Traditionally, cooled infrared material systems such as indium antimonide, platinum silicide, mercury cadmium telluride, and arsenic doped silicon (Si:As) have dominated infrared detection. Improvement in surveillance sensors and interceptor seekers requires large size, highly uniform, and multicolor IR focal plane arrays involving medium wave, long wave, and very long wave IR regions. Among the competing technologies are the quantum well infrared photodetectors based on lattice matched or strained III-V material systems. This paper discusses cooled IR technology with emphasis on QWIP and MCT. Details will be given concerning device physics, material growth, device fabrication, device performance, and cost effectiveness for LWIR, VLWIR, and multicolor focal plane array applications.

During the SOS response, LexA repressor is inactivated by specific cleavage. Although cleavage requires RecA protein in vivo, RecA acts indirectly as a coprotease by stimulating an inherent self-cleavage activity of LexA. In lambda lysogens, cleavage of lambda Cl repressor in a similar but far slower reaction results in prophage induction. We describe an intermolecular cleavage reaction in which the C-terminal fragment of LexA acted as an enzyme to cleave other molecules of LexA. The C-terminal fragment of lambda repressor cleaved the LexA substrates about as efficiently as did the LexA enzyme, suggesting that the slow rate of Cl self-cleavage results from a weak interaction between its cleavage site and the active site.

The CI repressor protein from the temperate lambdoid phage HK022 was purified to near homogeneity and used in DNase I footprinting analyses to identify six binding sites in this phage. All these sites contained homologous 15 bp inverted repeats. Three of these 15 bp inverted repeats were located between the cI and cro (OR1 to OR3), and the other three were 3′ to the cI gene (OL1 to OL3). Two of these sites were identified as operator sites for the repressor by DNA sequence analyses of virulent phage mutants. Almost all these mutations identified lay within the 15 bp inverted repeats comprising OR1 and OR2, and almost all were in the most highly conserved positions in the operators. The majority of virulent mutants contained mutations in both OR1 and OR2. Intrinsic affinities for individual operators were measured by DNase I footprinting analyses using DNA fragments which contained a single wild-type operator adjacent to two mutant operators. Comparison of these values with the affinity observed with these sites in the wild-type operator indicated that HK022 CI repressor bound cooperatively to OR1 and OR2 with a cooperativity parameter, ω, of almost 2000. Cooperative binding occurred in an alternative pairwise fashion, as previously seen with γ CI repressor. In addition to cooperative binding between two adjacent operators, the repressor also increased the affinity for adjacent non-specific DNA sites, resulting in a periodic pattern of binding termed "phasing". This phasing pattern extended beyond regions predicted for pair-wise interaction, but was significantly decreased on a template with two adjacent operators, suggesting that pairwise cooperativity interfered with phasing.

We have studied the effects of AlGaAs energy barriers surrounding self-assembled InAs quantum dots in a GaAs matrix on the properties of solar cells made with multiple quantum dot layers in the active region of a photodiode. We have compared the fenced dot samples with conventional InAs/GaAs quantum dot solar cells and with GaAs reference cells. We have found that, contrary to theoretical predictions, the AlGaAs fence layers do not enhance the transport properties of photogenerated carriers but instead suppress the extraction of the carriers excited in the dots by light with wavelengths longer than the cutoff wavelength of the GaAs matrix material. Both the standard quantum dots and the fenced dots were found to give solar cell performance comparable to the GaAs reference cells for certain active region thicknesses but neither showed enhancement due to the longer wavelength absorption or improved carrier transport.

Many specific DNA-binding proteins bind to sites with dyad symmetry, and the bound form of the protein is a dimer. For some proteins, dimers form in solution and bind to DNA. LexA repressor of Escherichia coli has been used to test an alternative binding model in which two monomers bind sequentially. This model predicts that a repressor monomer should bind with high specificity to an isolated operator half-site. Monomer binding to a half-site was observed. A second monomer bound to an intact operator far more tightly than the first monomer; this cooperativity arose from protein-protein contacts.

In saturation mutagenesis of a protein, pools of DNA molecules are made containing a mixture of codons at a specific position. In cases where genetic methods allow screens or selections for altered function, a background of nonsense mutations can complicate genetic analysis of the resulting mutations. Methods are proposed for elimination of those molecules containing stop codons at the target codon from the pool, and for identifying positions to which these methods may be applied. Application of these methods should ensure that all changes are missense mutations, thereby simplifying genetic analysis.

Crosses between various amber mutants of phage λ were carried out in thymine-requiring hosts in growth medium containing thymine or 5-bromouracil (BU). When BU replaced thymine, either in the parental phage DNA and/or in the medium (and presumably therefore in progeny DNA), the fraction of recombinants among the progeny was enhanced several-fold. This enhancement was due, at least in part, to an increase in the number of rounds of mating. A similar enhancement resulted from a period of thymine starvation.

The SOS regulatory system of E. coli controls the cellular response to DNA damage and other treatments which interfere with normal DNA replication. This system can exist in two states--a repressed state, in which a set of genes (SOS genes) is repressed by the LexA repressor; and an induced state, in which the RecA protein is activated as a specific protease which cleaves LexA repressor, leading to derepression of the SOS genes. This article reviews evidence that the state of the SOS regulatory system is controlled by the level of RecA protease activity. This level is controlled in turn by a reversible activation by one or more cofactors. In vitro studies indicate that ATP or dATP and single-stranded polynucleotide are both required to activate the protease; the identity of the in vivo cofactors ("inducing signals") is not yet certain. New experiments are also described which characterize the in vivo cleavage of LexA repressor. These data support the model that the level of RecA protease controls the state of the regulatory system.

LexA repressor of Escherichia coli and phage lambda repressor are inactivated in vivo and in vitro by specific cleavage of an Ala-Gly peptide bond in reactions requiring RecA protein. At mildly alkaline pH, the in vitro cleavage reaction also proceeds spontaneously, suggesting that peptide bond hydrolysis is an activity of the repressors rather than of RecA. The spontaneous cleavage reaction, termed "autodigestion", has been characterized for the LexA and lambda repressors. The results show that the reaction is intramolecular. The rate of LexA autodigestion was studied over the pH range 7.15-11.77 and over the temperature range 4-46 degrees C. The logarithm of the rate constant increased linearly with pH and reached a plateau value (2.5 X 10(-3) s-1 at 37 degrees C) at pH above 10. The data closely followed a model in which a single residue side chain (apparent pK = 9.8 at 37 degrees C) must be deprotonated for the protein to show activity. Analysis of the temperature dependence gave the heat of proton dissociation as 19.9 kcal/mol and the heat of activation for hydrolysis as 15.3 kcal/mol at 25 degrees C. Autodigestion of lambda repressor, studied over the pH range 8.65-10.70 at 37 degrees C, was similar to the LexA reaction in its pH dependence, yielding a pK of 9.8. The maximum rate at 37 degrees C for lambda repressor, 6.1 X 10(-5) s-1, was 40 times slower than for LexA, a difference similar to that previously observed in vivo and in vitro for RecA-dependent cleavage reactions. There was no significant solvent deuterium isotope effect on the autodigestion of LexA. Changes in buffer composition, including high concentrations of glycine for lambda repressor and of imidazole or hydroxylamine for LexA, indicated that solvent components other than water do not participate in the rate-determining step. Removal or addition of metal ions did not significantly affect LexA autodigestion. These and other observations suggest that the deprotonated form of an amino acid side chain plays a central role in the chemistry of the cleavage reaction. The above observations establish repressor autodigestion as a member of an emerging set of biologically important self-processing reactions.

Certain treatments that damage DNA and/or inhibit replication in E. coli have been reported to induce synthesis of a new protein, termed protein X, in recA+ lexA+ strains. We have examined some of the treatments that might induce protein X and we have, in particular, tested the hypothesis of Gudas and Pardee (1975) that DNA degradation products play an essential role in the induction process. We confirmed that UV irradiation, nalidixic acid treatment, or thymine starvation result in protein X synthesis in wild type strains. However, we found that UV irradiation, unlike nalidixic acid, also induced protein X in recB strains, in which little DNA degradation occurs. Furthermore, we found that the presence of DNA fragments resulting from host-controlled restriction of phage lambda DNA did not affect protein X synthesis. We conclude that no causal relationship exists between the production of DNA fragments and induction of protein X. The presence of the plasmid R46, which confers enhanced mutagenesis and UV resistance on its host, did not affect protein X synthesis. Growth in the presence of 5-bromouracil, which does not result in production of degradation fragments, resulted eventually in a low rate of protein X synthesis. In dnaA mutants, deficient in the initiation of new rounds of replication, UV irradiation induced protein X, again unlike nalidixic acid. Thus, the inhibition of active replication forks is not an essential requirement for protein X induction.

λ exonuclease exhibits a much greater rate of attack on native deoxyribonucleic acid than on denatured DNA, the difference in rate being 350-fold for T7 phage DNA. The attack is exclusively exonucleolytic, and the only acid-soluble products are deoxyribonucleoside 5‘-monophosphates. In contrast to the known Escherichia coli exonucleases, its initial site of attack is at the 5‘ termini of a DNA molecule, with a strong preference for termini bearing a phosphoryl residue as compared with those bearing a free hydroxyl group.

The deoxyribonucleic acid exonuclease induced by bacteriophage λ has been obtained in crystalline and physically homogeneous form. The purified preparation is free of detectable contamination by the endonuclease I, DNA polymerase, DNA phosphatase-exonuclease, alkaline phosphatase, 5'-nucleotidase, and ribonuclease activities known to be present in Escherichia coli; it may contain a trace (0.01%) of exonuclease I.

A scheme is given for the rapid preparation of NAD specifically labeled at a very high specific radioactivity with 3H at the 4-position of the nicotinamide ring.

We have observed an intensity‐dependent shift in the exciton absorption features for a system of coupled quantum wells in which a thin barrier separates two wells of unequal widths. We found that for a fixed external bias on the sample, the normal quantum Stark shift of the excitons in the wells was suppressed when light of 1 μW/cm2 intensity was used to measure the absorption. The shift became systematically larger as the light intensity was decreased to as low as 100 pW/cm2. This extremely low‐level nonlinearity may be due to internal fields that arise when free electrons and holes are separated within the coupled‐well system.

We have found that the uvrB gene of Escherichia coli is transcribed from at least two promoters, which we call P1 and P2. Transcription from P1 begins with an ATP at +1, and transcription from P2 begins primarily with a GTP at -31. A binding site for the lexA protein (LEXA), located between the -35 sequence and Pribnow box of P2, regulates transcription from this promoter. In vitro, LEXA inhibits transcription from P2 but has no detectable effect on transcription from P1. A third promoter, P3, was also detected at -341; transcription from P3 is toward uvrB but terminates in vitro in the region of the LEXA binding site. The binding of LEXA to P2 inhibits transcription from the P3 promoter even though several hundred nucleotides separate the two promoters. The data suggest that a transcribing RNA polymerase stalls when it reaches the repressor-operator complex but remains bound to the DNA, causing a jamming of RNA polymerases between P3 and the repressor-operator complex at P2. The physiological significance of P3 is unknown.

Expression of phage lytic functions in λ lysogens of Escherichia coli is prevented by phage repressor, which binds to specific operator sequences on the prophage DNA. Phage induction accompanies the expression of an entire set of ‘SOS functions’, which occur when host DNA is damaged or DNA synthesis is interrupted. In these conditions, λ repressor is thought to be inactivated by recA protease, thereby leading to expression of phage lytic functions1. We have now characterized the repressor from a λ mutant, inds-1, which, as prophage, is more easily induced than wild-type phage by a weak inducing treatment2. At high concentrations, λinds-1 repressor differs from wild-type repressor in two ways: whereas wild-type repressor forms dimers and becomes relatively resistant to the recA protease, a much greater proportion of λinds-1 repressor remains monomeric and the protein remains sensitive to the protease. These findings support previous indications3 that repressor monomers are the preferred substrate for protease, both in vivo and in vitro.

Treatment of Escherichia coli with radiation or chemical carcinogens induces a number of cellular functions to counteract the damage inflicted by such treatments (see ref. 1 for a review). One of these inducible functions is the SOS response, which is controlled by the recA and lexA genes: single-stranded DNA produced as a result of DNA damage binds to recA protein (RecA), activating its protease function; activated RecA cuts lexA protein (LexA), the represser of the SOS genes, thereby inactivating it and turning on these genes2–4. The SOS genes include recA and lexA themselves, and recent work has shown that the two genes have similar operators where LexA binds in vitro, inhibiting their transcription3,4. It is anticipated that other genes under the control of recA and lexA will have similar operators. Recent genetic5,6 and biochemical7 data suggest that the excision repair genes uvrA and uvrB are part of the recA–lexA regulon and are at least partly responsible for recA–lexA-mediated inducible DNA repair. In support of these results we have reported that uvrB has a promoter that is repressed by LexA in vitro8. We now show that uvrA has an operator similar to those of other SOS genes and that LexA binds to this operator and inhibits the transcription of the uvrA gene in vitro.

The product of bacteriophage T4 gene 32, DNA-unwinding protein or 32 protein, is required in large amounts for normal phage growth. Gene 32 amber mutants are unable to form plaques on ochre-suppressing hosts, presumably because there is too little 32 protein for efficient growth. However, variants arise which can form small, turbid plaques on these hosts. These variants contain a second mutation, termed sud , which maps between genes 39 and 56, far from gene 32. Sud mutants are recessive to wildtype. By themselves, sud mutants exhibit a short delay in the onset of rapid DNA synthesis relative to wild-type. The am A453 (gene 32)- sud double mutants exhibit a very long delay in DNA synthesis relative to wild-type when infecting the ochre-suppressing host, after which DNA synthesis proceeds at the same rate as shown by wild-type, while am A453 synthesizes DNA at a rate 30% that shown by wild-type after a short delay. Moreover, am A453 converts the DNA made to mature progeny much less efficiently than does wild-type, whereas one of the sud mutants described resembles wild-type more closely in this respect. The possible meaning of these observations is discussed.

Recent work in this" 2 and other. laboratories3-6 has demonstrated the existence of enzymes which catalyze the covalent closure of single-strand breaks in duplex DNA. Interest in such enzymes derives in part from their probable biological importance; thus, the enzyme induced by infection with bacteriophage T4 has been shown to be necessary for multiplication of the phage.7 Characterization of purified enzymes from uninfected2' 4 5 and T4-infected3 Escherichia coli has revealed a requirement in each case for a nucleotide cofactor. The enzyme from uninfected cells specifically requires DPN,2 I which is broken down into AMP and NMN in the course of the reaction; the enzyme from T4infected cells, by contrast, splits ATP into AMP and PPi.3 In both cases, 3'-5' plhosphodiester bonds are formed by covalent joining of the two apposed DNA termini at a single-strand break. This class of enzymes thus catalyzes a complex reaction which may be expected to proceed in several distinguishable stages. This report describes the first step of the reaction catalyzed by the DNA ligase of uninfected E. coli. Upon admixture with DPN in the absence of DNA, the enzyme forms a stable enzyme-adenylate complex, releasing NMN. The enzyme-adenylate complex, as isolated by gel filtration, can seal single-strand breaks in DNA in the absence of DPN. AMP is released in this reaction. The enzyme-adenylate complex may also be discharged with NMN to yield DPN by reversal of the reaction which formed the complex. As a consequence an exchange between DPN and NMN can be observed, as noted by Olivera and Lehman.5 These reactions are summarized in the following equations

Publisher Summary Lambda exonuclease is discovered and partially purified using induced cultures of E. coli K12 (λ + ). Under the conditions of induction and subsequent growth described in this chapter, the specific enzymatic activity of a crude extract made from this strain is thirty times greater than that observed in a crude extract of W3101 (λ + )/1,5 made 70 minutes after UV induction. The λ exonuclease shows a strong preference for native DNA. The rate of attack on native E. coli DNA is 50-250 times greater than that on denatured E. coli DNA, the ratio depending on the preparation of DNA used. The λ exonuclease releases 5'-mononucleotides as acid-soluble digestion products of DNA. The acid-soluble material cochromatographs with authentic deoxyribonueleoside 5'-monophosphates; in a digest of 32 P-labeled DNA, the acid-soluble 32 p is completely susceptible to the action of 5'-nucleotidase from snake venom. The enzyme initiates its attack on DNA at the 5'-terminus of a DNA strand. When phage λ DNA is labeled specifically at its 3'-termini with α- 32 P-labeled deoxyribonucleoside triphosphates by E. coli DNA polymerase and used as substrate, exonuelease catalyzes a very slow release of 32 P relative to total nucleotide, while exonuclease III liberates all the 32 P before detectable amounts of total nucleotide are released.