On the Nature of CIS-Acting Regulatory Proteins and Genetic Organization in Bacteriophage: The Example of Gene Q of Bacteriophage λ

We note the existence of a "partially cis-acting" regulatory protein of bacteriophage λ: the product of the phage Q gene. We suggest that there may be a complete spectrum from "all cis" to "all trans" for such regulatory proteins. This behavior might arise because a DNA-binding protein either acts at a nearby (cis) site soon after synthesis or becomes "lost" for its trans activity on another genome through nonspecific interactions with DNA. Our proposed explanation provides one evolutionary basis for the linkage of genes for regulatory proteins and the sites at which such proteins act; it also suggests a possible rationale for the "metabolic instability" of certain regulatory proteins.     

Preparation of Plasmid λdv from Bacteriophage λ: Role of Promoter-Operator in the Plasmid Replicon

A technique has been described for selection of bacteria carrying plasmid lambdadv. With this technique, the effects of mutations in the promoter-operators were compared on the production and perpetuation of the plasmid. It was found that "left" promoter-operator that controls leftward gene expressions can be deleted from the plasmid genome. Some mutations of "right" promoter-operator (pRoR) that controls expression of genes tof, O, and P affect the stability of the plasmid. However, the plasmid genome accomodates a variety of pRoR mutations within a reasonable but different degree of constitutivity. Some new promoter mutations that allow bypass of the pRoR cannot be carried in the plasmid genome. From these findings it was proposed that the plasmid replicon has one indispensable promoter-operator that controls expression of all the genes related to its own replication, although a variety of constitutive mutations can be accommodated in the pRorR.     

Polar Mutations in the Left Arm of Bacteriophage λi434

By studying complementation between frameshift and nonsense mutants located in the structural genes for the head of bacteriophage lambdai434, we found mutations in gene B which are polar on genes C and D and one mutation in gene E which is polar on gene F.     

TRIM30α Is a Negative-Feedback Regulator of the Intracellular DNA and DNA Virus-Triggered Response by Targeting STING

Uncontrolled immune responses to intracellular DNA have been shown to induce autoimmune diseases. Homeostasis regulation of immune responses to cytosolic DNA is critical for limiting the risk of autoimmunity and survival of the host. Here, we report that the E3 ubiquitin ligase tripartite motif protein 30α (TRIM30α) was induced by herpes simplex virus type 1 (HSV-1) infection in dendritic cells (DCs). Knockdown or genetic ablation of TRIM30α augmented the type I IFNs and interleukin-6 response to intracellular DNA and DNA viruses. Trim30α-deficient mice were more resistant to infection by DNA viruses. Biochemical analyses showed that TRIM30α interacted with the stimulator of interferon genes (STING), which is a critical regulator of the DNA-sensing response. Overexpression of TRIM30α promoted the degradation of STING via K48-linked ubiquitination at Lys275 through a proteasome-dependent pathway. These findings indicate that E3 ligase TRIM30α is an important negative-feedback regulator of innate immune responses to DNA viruses by targeting STING.     

Low-Frequency Rescue of a Genetic Marker in Deoxyribonucleic Acid from Bacillus Bacteriophage φ105 by Superinfecting Bacteriophage

Markers in gene L, which maps at the right end of the vegetative and prophage maps, are rescued at a strongly reduced frequency from mature 105 deoxyribonucleic acid (DNA) by superinfecting phage but at high frequency from vegetative and prophage DNA. It is suggested that the ends of mature DNA are degraded when DNA is taken up by competent cells.     

What Is the Primary Cause of Individual Differences in Contrast Sensitivity?

One of the primary objectives of early visual processing is the detection of luminance variations, often termed image contrast. Normal observers can differ in this ability by at least a factor of 4, yet this variation is typically overlooked, and has never been convincingly explained. This study uses two techniques to investigate the main source of individual variations in contrast sensitivity. First, a noise masking experiment assessed whether differences were due to the observer’s internal noise, or the efficiency with which they extracted information from the stimulus. Second, contrast discrimination functions from 18 previous studies were compared (pairwise, within studies) using a computational model to determine whether differences were due to internal noise or the low level gain properties of contrast transduction. Taken together, the evidence points to differences in contrast gain as being responsible for the majority of individual variation across the normal population. This result is compared with related findings in attention and amblyopia.     

Crystal Structure of the Bacteriophage Qβ Coat Protein in Complex with the RNA Operator of the Replicase Gene

The coat proteins of single-stranded RNA bacteriophages specifically recognize and bind to a hairpin structure in their genome at the beginning of the replicase gene. The interaction serves to repress the synthesis of the replicase enzyme late in infection and contributes to the specific encapsidation of phage RNA. While this mechanism is conserved throughout the Leviviridae family, the coat protein and operator sequences from different phages show remarkable variation, serving as prime examples for the co-evolution of protein and RNA structure. To better understand the protein–RNA interactions in this virus family, we have determined the three-dimensional structure of the coat protein from bacteriophage Qβ bound to its cognate translational operator. The RNA binding mode of Qβ coat protein shares several features with that of the widely studied phage MS2, but only one nucleotide base in the hairpin loop makes sequence-specific contacts with the protein. Unlike in other RNA phages, the Qβ coat protein does not utilize an adenine-recognition pocket for binding a bulged adenine base in the hairpin stem but instead uses a stacking interaction with a tyrosine side chain to accommodate the base. The extended loop between β strands E and F of Qβ coat protein makes contacts with the lower part of the RNA stem, explaining the greater length dependence of the RNA helix for optimal binding to the protein. Consequently, the complex structure allows the proposal of a mechanism by which the Qβ coat protein recognizes and discriminates in favor of its cognate RNA.     

Enhancement of bacteriophage λ stability using a λQ − S − mutant in the continuous culture of Escherichia coli

In this study, we used a bacteriophage λQ ⁻ S ⁻ mutant that increased the stability of recombinant Escherichia coli during continuous culture. The operation was conducted in two stages: the first stage was carried out to promote cell growth, and the second stage was performed for product formation. The productivity of recombinant proteins depends on the substrate concentration of the fresh medium supplied to the second stage (S ₃) and dilution rate of the second stage (D ₂). With the optimal value of S ₃ and D ₂, the first and second stages were stably maintained for 170 and 80 h, respectively. To further improve this process, a three-stage continuous process was conducted with an additional induction stage between the growth and production stages. Compared with the two-stage operation, the stable production period was extended by 1.7 fold, and the recombinant protein production increased by 1.3 fold.     

S-Glutathionylation of the Na,K-ATPase Catalytic α Subunit Is a Determinant of the Enzyme Redox Sensitivity

Na,K-ATPase is highly sensitive to changes in the redox state, and yet the mechanisms of its redox sensitivity remain unclear. We have explored the possible involvement of S-glutathionylation of the catalytic α subunit in redox-induced responses. For the first time, the presence of S-glutathionylated cysteine residues was shown in the α subunit in duck salt glands, rabbit kidneys, and rat myocardium. Exposure of the Na,K-ATPase to oxidized glutathione (GSSG) resulted in an increase in the number of S-glutathionylated cysteine residues. Increase in S-glutathionylation was associated with dose- and time-dependent suppression of the enzyme function up to its complete inhibition. The enzyme inhibition concurred with S-glutathionylation of the Cys-454, -458, -459, and -244. Upon binding of glutathione to these cysteines, the enzyme was unable to interact with adenine nucleotides. Inhibition of the Na,K-ATPase by GSSG did not occur in the presence of ATP at concentrations above 0.5 mm. Deglutathionylation of the α subunit catalyzed by glutaredoxin or dithiothreitol resulted in restoration of the Na,K-ATPase activity. Oxidation of regulatory cysteines made them inaccessible for glutathionylation but had no profound effect on the enzyme activity. Regulatory S-glutathionylation of the α subunit was induced in rat myocardium in response to hypoxia and was associated with oxidative stress and ATP depletion. S-Glutathionylation was followed by suppression of the Na,K-ATPase activity. The rat α2 isoform was more sensitive to GSSG than the α1 isoform. Our findings imply that regulatory S-glutathionylation of the catalytic subunit plays a key role in the redox-induced regulation of Na,K-ATPase activity.     

Characteristics of φT, the Temperate Bacteriophage Carried by Bacillus megaterium 899a

Phage T was the only phage observed in lysates of Bacillus megaterium 899a induced with mitomycin C, 0.35 mug/ml. The phage adsorbed slowly to its host in nutrient agar, giving rise to plaques of varying sizes and turbidity. Only clear plaques were observed when the phage and host cells were preincubated in an adsorption buffer and plated under optimum conditions. Plaque turbidity was caused by either the addition of 0.5 x 10(-2) to 1.0 x 10(-2) M CaCl(2) to the phage assay medium, or by raising the incubation temperature to 34 C. Phage T purified on a CsCl gradient had a density of 1.48 g/ml in CsCl and the extracted phage DNA had a buoyant density in CsCl of 1.6975 g/ml, equivalent to 38.2% guanine plus cytosine. The phage was rapidly inactivated at 75 C and was unstable in the presence of chloroform at 4 C, but it was stable in buffer stored in ice. When stage I sporulating cells were induced with mitomycin C, phage were carried into spores which when germinated lyse with the release of phi T. The burst size on induction of early-log vegetative cells was 52, whereas the burst size of induced T(0) sporulating cells, diluted in fresh medium, was 47 for a sporulating strain and 140 for an asporogenous mutant. A typical phage T had a long, noncontracting tail 240 nm long, 9 to 11 nm wide, with a repeating disk unit along the tail, 4 nm in size center to center. The tail ended in a small disk (15 nm wide) which is presumably for attachment to the host. The hexagonal head measures 68 by 57 nm and is composed of donut-shaped units 9 nm in diameter.     

Role of Toxin ζ and Starvation Responses in the Sensitivity to Antimicrobials

A fraction of otherwise antimicrobial-sensitive Bacillus subtilis cells, called persisters, are phenotypically tolerant of antimicrobial treatment. We report that, independently of B. subtilis'' growth phase, transient ζ toxin expression induces a dormant state and alters cellular responses so that cells are more sensitive to antimicrobials with different modes of action. This outcome is modulated by fine tuning (p)ppGpp and GTP levels: i) in the presence of low “dysregulated” (p)ppGpp levels (as in relA⁻ cells) hyper-tolerance to both toxin and antimicrobials was observed; ii) physiological or low (p)ppGpp levels (as in the wild-type, sasA⁻, sasB⁻ and relA⁻sasA⁻ context) show a normal toxin and antimicrobial tolerance; and iii) lower levels (in relA⁻sasB⁻) or absence of (p)ppGpp (in the relA⁻sasA⁻sasB⁻ context), in concert with elevated GTP levels, potentiate the efficacy of both toxin and antimicrobial action, rendering tolerance vulnerable to eradication.     

DNA Synthesis at the Site of a Red-Mediated Exchange in Phage λ

In phage lambda, progeny particles bearing unreplicated chromosomes are recombinant by action of lambda's Red system only near the right end of the chromosome. These recombinants are frequently heterozygous (heteroduplex) for markers located there. In replication-blocked crosses involving two heavy-labeled parents we find that particles in the solitary peak, containing progeny with fully conserved DNA, vary in density. Those on the heavy side of this peak are more apt to be heterozygous than are those on the light side. The data fit a model in which a double chain cut at cos, lambda's packaging origin, is followed by partial exonucleolytic degradation of lambda's r chain from the right end leftward. The exposed l chain, which thereby constitutes a 3' overhang, invades an intact, circular homologue after itself suffering some degradation. Completion of the recombinant chromosome sometimes involves DNA synthesis primed by the invading chain.     

Electrophoretic and Other Properties of Bacteriophage Qβ: the Effect of a Variable Number of Read-Through Proteins

When subjected to electrophoresis in polyacrylamide gels, the virions of wild-type Qbeta bacteriophage are found in a single, major, anomalously wide band. With Qbeta mutant 27-2, this wide band is replaced by a set of narrow, well-defined bands. The most rapidly migrating band of the mutant, comprising less than 10% of the total, contains defective virions. These virions have sedimentation coefficients ranging from 70 to 100% of the bulk of the unfractionated mutant, they contain no read-through protein (protein IIb), and they are deficient in maturation protein and contain fragmented RNA. The second band, comprising less than 3% of the total virus, has not been well characterized. The virions in the remaining electrophoretic bands are infective. Their distribution into bands is believed due to differences in their effective volume resulting from differences in their content of protein IIb. The most rapidly migrating band of this series contains virions with a few molecules of IIb protein, whereas the more slowly migrating bands contain virions with a larger number of IIb molecules. The adjacent bands in the series contain virions which differ by approximately three IIb molecules. Wild-type Qbeta virus is similar to the mutant in that the more slowly migrating virions contain more protein IIb than the more rapidly migrating virions. Their failure to resolve into distinct bands upon electrophoresis is believed due to a less restricted packing of protein IIb into their virions. Both wild-type Qbeta and mutant 27-2 also have 1 to 5% of the virions in the form of dimers which migrate with approximately one-half the mobility of the respective monomer forms. When the average amount of IIb per virion is increased by growth of the virus in a UGA suppressor strain, the electrophoretic pattern is altered. In the case of wild-type Qbeta, the single band is wider, whereas with Qbeta mutant 27-2 there occurs an increased number of partially resolved narrow bands. We suggest that the structural feature responsible for the difference in electrophoretic pattern between mutant 27-2 and wild-type Qbeta is the mode of IIb packing in the virions. In the mutant, the IIb proteins are found in the virions only in multiples of three, whereas wild-type virions may differ by only a single IIb protein.     

Seizure Sensitivity Is Ameliorated by Targeted Expression of K+–Cl? Cotransporter Function in the Mushroom Body of the Drosophila Brain

The kcc^DHS1 allele of kazachoc (kcc) was identified as a seizure-enhancer mutation exacerbating the bang-sensitive (BS) paralytic behavioral phenotypes of several seizure-sensitive Drosophila mutants. On their own, young kcc^DHS1 flies also display seizure-like behavior and demonstrate a reduced threshold for seizures induced by electroconvulsive shock. The product of kcc shows substantial homology to KCC2, the mammalian neuronal K⁺–Cl⁻ cotransporter. The kcc^DHS1 allele is a hypomorph, and its seizure-like phenotype reflects reduced expression of the kcc gene. We report here that kcc functions as a K⁺–Cl⁻ cotransporter when expressed heterologously in Xenopus laevis oocytes: under hypotonic conditions that induce oocyte swelling, oocytes that express Drosophila kcc display robust ion transport activity observed as a Cl⁻-dependent uptake of the K⁺ congener ⁸⁶Rb⁺. Ectopic, spatially restricted expression of a UAS-kcc⁺ transgene was used to determine where cotransporter function is required in order to rescue the kcc^DHS1 BS paralytic phenotype. Interestingly, phenotypic rescue is largely accounted for by targeted, circumscribed expression in the mushroom bodies (MBs) and the ellipsoid body (EB) of the central complex. Intriguingly, we observed that MB induction of kcc⁺ functioned as a general seizure suppressor in Drosophila. Drosophila MBs have generated considerable interest especially for their role as the neural substrate for olfactory learning and memory; they have not been previously implicated in seizure susceptibility. We show that kcc^DHS1 seizure sensitivity in MB neurons acts via a weakening of chemical synaptic inhibition by GABAergic transmission and suggest that this is due to disruption of intracellular Cl⁻ gradients in MB neurons.Mushroom body (MB) expression of the kazachoc (kcc) K⁺–Cl⁻ cotransporter is shown here to rescue seizure-sensitive phenotypes in Drosophila through an effect on GABAergic fast synaptic inhibition. Heretofore, considerable interest has focused on the MB because of its essential role in olfactory learning and memory (Heisenberg 2003; Davis 2005; Keene and Waddell 2007; Berry et al. 2008). The MB occupies a central position in the fly nervous system, integrating incoming olfactory, mechanical, taste, and visual sensory signals and then sorting the distribution of outgoing motor signals (Heisenberg 2003). Short- and long-term alteration of individual nerve cell physiology in the MB is thought to form the basis of learning and memory (Davis 2005; Keene and Waddell 2007; Berry et al. 2008). A role for the MB in seizure susceptibility has not previously been suspected. Here we suggest that the orderly arrangements of axons and neuropile of MB Kenyon cells (KCs) not only facilitate learning and memory, but also provide the type of anatomical substrate in flies that is thought to be essential for seizure spread in the mammalian brain (Hauser and Hesdorffer 1990; Traub and Miles 1991).Inhibitory synaptic transmission in Drosophila is thought to be mediated primarily by GABAergic neurons found throughout the CNS at all stages of development (Buchner et al. 1988; Jackson et al. 1990; Harrison et al. 1996; Yasuyama et al. 2002). γ-aminobutyric acid (GABA) is synthesized from glutamate via a conserved glutamic acid decarboxylase encoded by the Drosophila Gad1 gene (Jackson et al. 1990; Buchner 1991). GABAergic activity is limited by sequestering extracellular GABA back into presynaptic neurons by GABA transporters that are sensitive to inhibition by dl-2,4-diaminobutyric acid, nipecotic acid, and valproic acid (Neckameyer and Cooper 1998; Leal et al. 2004). Three ionotropic GABA_A receptor subunits have been identified in Drosophila and are encoded by the Rdl, LCCH3, and GRD loci (Hosie et al. 1997). When expressed heterotopically in Xenopus oocytes, the best studied of these, Rdl, forms GABA-gated Cl⁻ channels that are sensitive to block by picrotoxin (ffrench-Constant et al. 1991, 1993; Zhang et al. 1995). Inhibitory Cl⁻ currents are dependent on maintenance of Cl⁻ gradients, particularly in low intracellular Cl⁻ concentrations. In the fly, Cl⁻ gradients appear to be maintained by the kcc K⁺–Cl⁻ cotransporter (Hekmat-Scafe et al. 2006).Chemical synaptic transmission onto MB neurons has been examined in dissociated KCs in primary culture (Su and O''dowd 2003). Spontaneous miniature excitatory postsynaptic currents (EPSCs) are mediated mainly by nicotinic acetylcholine (ACh) receptors. Miniature inhibitory postsynaptic currents (IPSCs) appear to be mediated primarily by picrotoxin-sensitive GABA_A receptors, probably encoded by Rdl (Su and O''dowd 2003; Harrison et al. 1996). In vivo, cholinergic inputs to the MB are thought to arise primarily from antennal lobe projection neurons (Yasuyama et al. 2002). Two antennal lobe neurons that project to the MB, the anterior paired lateral (APL) neurons, were recently shown to be GABAergic (Liu and Davis, 2009). Additional GABAergic inputs to the Drosophila MB seem likely; in locust they appear to come from a poorly understood region of the brain called the lateral horn, which is itself also driven by antennal lobe projection neurons (Perez-Orive et al. 2002).Previously, we identified the kcc^DHS1 partial loss-of-function mutation as a seizure enhancer that also causes increased seizure sensitivity in young flies (Hekmat-Scafe et al. 2006). The kcc product shows homology to the mammalian KCC2 K⁺–Cl⁻ cotransporter, and we inferred that a decrease in inhibitory synaptic strength is responsible for causing the seizure phenotypes. In this study, we describe our search for identifying the source of these vulnerable inhibitory synapses and report that they appear to lie primarily in the MBs of the Drosophila brain. Further, we speculate on the possibility of their involvement in synaptic plasticity functions of the MB.     

CAROTENOID CLEAVAGE DIOXYGENASE4 Is a Negative Regulator of β-Carotene Content in Arabidopsis Seeds

Experimental approaches targeting carotenoid biosynthetic enzymes have successfully increased the seed β-carotene content of crops. However, linkage analysis of seed carotenoids in Arabidopsis thaliana recombinant inbred populations showed that only 21% of quantitative trait loci, including those for β-carotene, encode carotenoid biosynthetic enzymes in their intervals. Thus, numerous loci remain uncharacterized and underutilized in biofortification approaches. Linkage mapping and genome-wide association studies of Arabidopsis seed carotenoids identified CAROTENOID CLEAVAGE DIOXYGENASE4 (CCD4) as a major negative regulator of seed carotenoid content, especially β-carotene. Loss of CCD4 function did not affect carotenoid homeostasis during seed development but greatly reduced carotenoid degradation during seed desiccation, increasing β-carotene content 8.4-fold relative to the wild type. Allelic complementation of a ccd4 null mutant demonstrated that single-nucleotide polymorphisms and insertions and deletions at the locus affect dry seed carotenoid content, due at least partly to differences in CCD4 expression. CCD4 also plays a major role in carotenoid turnover during dark-induced leaf senescence, with β-carotene accumulation again most strongly affected in the ccd4 mutant. These results demonstrate that CCD4 plays a major role in β-carotene degradation in drying seeds and senescing leaves and suggest that CCD4 orthologs would be promising targets for stabilizing and increasing the level of provitamin A carotenoids in seeds of major food crops.