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Non-mammalian vertebrates have an intrinsically photosensitive iris and thus a local pupillary light reflex (PLR). In contrast, it is thought that the PLR in mammals generally requires neuronal circuitry connecting the eye and the brain. Here we report that an intrinsic component of the PLR is in fact widespread in nocturnal and crepuscular mammals. In mouse, this intrinsic PLR requires the visual pigment melanopsin; it also requires PLCβ4, a vertebrate homologue of the Drosophila Norp A phospholipase C which mediates rhabdomeric phototransduction. The Plcb4^(−/−) genotype, in addition to removing the intrinsic PLR, also essentially eliminates the intrinsic light response of the M1 subtype of melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (M1-ipRGCs), which are by far the most photosensitive ipRGC subtype and also have the largest response to light. Ablating in mouse the expression of both TRPC6 and TRPC7, members of the TRP channel superfamily, also essentially eliminated the M1-ipRGC light response but the intrinsic PLR was not affected. Thus, melanopsin signalling exists in both iris and retina, involving a PLCβ4-mediated pathway that nonetheless diverges in the two locations.

Platelets are small disc-shaped cell fragments which undergo a rapid transformation when they encounter vascular damage. They become more spherical and extrude pseudopodia, their fibrinogen receptors are activated, causing them to aggregate, they release their granule contents, and eventually form a plug which is responsible for primary haemostasis. Activation of platelets is also implicated in the pathogenesis of unstable angina, myocardial infarction and stroke. Here we show that platelets from mice deficient in the α-subunit of the heterotrimeric guanine-nucleotide-binding protein Gq are unresponsive to a variety of physiological platelet activators. As a result, G α_q-deficient mice have increased bleeding times and are protected from collagen and adrenaline-induced thromboembolism. We conclude that G α_q is essential for the signalling processes used by different platelet activators and that it cannot be replaced by G α_i or the βγ subunits of the heterotrimeric G proteins. G α_q may thus be a new target for drugs designed to block the activation of platelets.

The heterotrimeric G proteins mediate a variety of cellular processes by coupling transmembrane receptors to different effector molecules, including adenylyl cyclases and inositol-phospholipid-specific phospholipase C (PLC)1-3. Activation of adenylyl cyclases results in the production of cyclic AMP and activation of cAMP-dependent protein kinase (PKA). Phospholipase C catalyses the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdInsP2) to generate diacylglycerol and inositol-1,4,5-triphosphate (InsP2), leading to the activation of protein kinase C (PKC) and the mobilization of intracellular calcium. The various PLC isoforms appear to be activated by different receptors, and in some cases by different G-protein components. There are four well-characterized forms of PLC-beta and all of them are activated to various extents by the G alpha q family of G proteins. Specific activation of PLC isoforms beta 2 and beta 3 by G-protein beta gamma subunits has also been reported. Although it has been suggested that PLC activity might be modulated by the adenylyl cyclase pathway, no clear link has been established between the two pathways. Here we report that cAMP-dependent protein kinase specifically inhibits G beta gamma-activated PLC-beta 2 activity but not that of the G alpha-activated PLC isoforms, and that the effect of PKA is not mimicked by PKC isozymes. Furthermore, we show that PKA directly phosphorylates serine residues of the PLC-beta 2 protein both in vivo and in vitro. Our results provide an insight into the specificity and nature of the crosstalk between the two G-protein-coupled signal transduction pathways.

We have used surface plasmon resonance biosensor technology to monitor the assembly and dynamics of a signal transduction complex which controls chemotaxis in Escherichia coli. A quaternary complex formed which consisted of the response regulator CheY, the histidine protein kinase CheA, a coupling protein CheW and a membrane-bound chemoreceptor Tar. Using various experimental conditions and mutant proteins, we have shown that the complex dissociates under conditions that favour phosphorylation of CheY. Direct physical analysis of interactions among proteins in this signal transduction pathway provides evidence for a previously unrecognized binding interaction between the kinase and its substrate. This interaction may be important for enhancing substrate specificity and preventing 'crosstalk' with other systems. The approach is generally applicable to furthering our understanding of how signalling complexes transduce intracellular messages.

THE activation of heterotrimeric G proteins results in the exchange of GDP bound to the α-subunit for GTP and the subsequent dissociation of a complex of the β- and γ-subunits (Gβγ). The α -subunits of different G proteins interact with a variety of effectors, but less is known about the function of the free Gβγ complex. Gβγ has been implicated in the activation of a cardiac potassium channel, a retinal phospholipase A2 (ref. 9) and a specific receptor kinase, and in vitro reconstitution experiments indicate that the Gβγ complex can act with Gα subunit to modulate the activity of different isoforms of adenylyl cyclase11. Of two phospholipase activities that can be separated in extracts of HL-60 cells, purified Gβγ is found to activate one of them. Here we report that in co-transfection assays Gβγ subunits specifically activate the β2 and not the β1 isoform of phospholipase, which acts on phosphatidylinositol. We use transfection assays to show also that receptor-mediated release of Gβγ from G proteins that are sensitive to pertussis toxin can result in activation of the phospholipase. This effect may be the basis of the pertussis-toxin-sensitive phospholipase C activation seen in some cell systems (reviewed in refs 13 and 14).

The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells — B lymphocytes (the cells of the immune system) and cardiac myocytes — to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.

Arrestins are soluble cytoplasmic proteins that bind to G-protein-coupled receptors, thus switching off activation of the G protein and terminating the signalling pathway that triggers the cellular response. Although visual arrestin has been shown to quench the catalytic activity of photoexcited, phosphorylated rhodopsin in a reconstituted system, its role in the intact rod cell remains unclear because phosphorylation alone reduces the catalytic activity of rhodopsin. Here we have recorded photocurrents of rods from transgenic mice in which one or both copies of the arrestin gene were disrupted. Photoresponses were unaffected when arrestin expression was halved, indicating that arrestin binding is not rate limiting for recovery of the rod photoresponse, as it is in Drosophila . With arrestin absent, the flash response displayed a rapid partial recovery followed by a prolonged final phase. This behaviour indicates that an arrestin-independent mechanism initiates the quench of rhodopsin's catalytic activity and that arrestin completes the quench. The intensity dependence of the photoresponse in rods lacking arrestin further suggests that, although arrestin is required for normal signal termination, it does not participate directly in light adaptation.

Platelets are small disc-shaped cell fragments which undergo a rapid transformation when they encounter vascular damage. They become more spherical and extrude pseudopodia, their fibrinogen receptors are activated, causing them to aggregate, they release their granule contents, and eventually form a plug which is responsible for primary haemostasis. Activation of platelets is also implicated in the pathogenesis of unstable angina, myocardial infarction and stroke. Here we show that platelets from mice deficient in the alpha-subunit of the heterotrimeric guanine-nucleotide-binding protein Gq are unresponsive to a variety of physiological platelet activators. As a result, G alpha(q)-deficient mice have increased bleeding times and are protected from collagen and adrenaline-induced thromboembolism. We conclude that G alpha(q) is essential for the signalling processes used by different platelet activators and that it cannot be replaced by G alpha(i) or the beta gamma subunits of the heterotrimeric G proteins. G alpha(q) may thus be a new target for drugs designed to block the activation of platelets.

The activation of heterotrimeric G proteins results in the exchange of GDP bound to the alpha-subunit for GTP and the subsequent dissociation of a complex of the beta- and gamma-subunits (G beta gamma). The alpha-subunits of different G proteins interact with a variety of effectors, but less is known about the function of the free G beta gamma complex. G beta gamma has been implicated in the activation of a cardiac potassium channel, a retinal phospholipase A2 (ref. 9) and a specific receptor kinase, and in vitro reconstitution experiments indicate that the G beta gamma complex can act with G alpha subunit to modulate the activity of different isoforms of adenylyl cyclase. Of two phospholipase activities that can be separated in extracts of HL-60 cells, purified G beta gamma is found to activate one of them. Here we report that in co-transfection assays G beta gamma subunits specifically activate the beta 2 and not the beta 1 isoform of phospholipase, which acts on phosphatidylinositol. We use transfection assays to show also that receptor-mediated release of G beta gamma from G proteins that are sensitive to pertussis toxin can result in activation of the phospholipase. This effect may be the basis of the pertussis-toxin-sensitive phospholipase C activation seen in some cell systems (reviewed in refs 13 and 14).

Transmembrane receptors monitor the extracellular environment and transmit information about changes in specific ligand concentration to the cytoplasm. It is not clear how the message gets across the cell membrane, but one proposal is that ligand binding induces a transmembrane conformational change in the receptor. Writing in Science late last year, members of Kim and Koshland's laboratories reported the determination of the three-dimensional structure of the extracellular domain of Tar, the bacterial aspartate receptor, with and without bound ligand, the hope being that the structures would provide a clear image of the conformational changes involved in signalling. Unfortunately, however, the picture remains blurred.

Many biological processes depend on the function of proteins that detect changes in a cell's environment and transmit the information to the cytoplasm, for example, peptide hormone receptors. In Escherichia coli this class of proteins is exemplified by the sensory transducers (also called signalling proteins or methyl-accepting chemotaxis proteins) which have a central role in mediating chemotactic behaviour. The sensory transducers are the products of four genes: tsr, tar, tap and trg. Each transducer detects changes in the environmental concentration of one or a very few attractants: Tsr, serine; Tar, aspartate and maltose; Tap, unknown; and Trg, ribose and galactose. Tsr and Tar act directly as chemoreceptors for the amino acid attractants and signal changes in their degree of occupancy to the flagellar apparatus. Detection of these changes in occupancy is made possible as the transducers are methylated at multiple glutamate residues such that their level of methylation reflects the most recent chemoeffector concentration. Biochemical and genetic information concerning the serine transducer protein has been accumulating rapidly but little is known about the structure of the molecule. We present here the nucleotide sequence of the tsr gene of E. coli; the amino acid sequence derived from it suggests that the Tsr transducer protein has a relatively simple transmembrane structure that may place limits on the mechanisms available for the transmission of sensory information into the cell.

A cascade of protein phosphorylation, initiated by autophosphorylation of the CheA protein, may be important in the signal transduction pathway of bacterial chemotaxis. A proteolytic fragment of CheA cannot autophosphorylate, but can still transfer phosphate to proteins that generate excitation and adaptation signals. The site of CheA phosphorylation is His 48; mutants altered at this position are non-chemotactic. Similar mechanisms of transient protein phosphorylation and phosphoryl group transfer seem to be involved in processing sensory data and in activating specific gene expression.

Diphtheria toxin (DT), a 63,000-molecular weight soluble protein, is toxic to most mammalian cells. The mechanism of intoxication involves a step in which one part of the molecule inserts into a membrane, facilitating the transport of the enzymatic fragment of the protein into the cytoplasm1,2. This event requires an acidic environment and seems to occur at the membrane of an endocytic vesicle3,4. Different cell lines and species differ in their sensitivities to DT—this is due at least in part to differences in the number of DT surface receptors on the cells5, but may also arise from differences in the membrane transport step. It is generally recognized that protein–lipid interactions are of critical importance in membrane transport phenomena (see ref. 6). Thus, it is of interest to determine whether the interaction of DT with membranes is modulated by their composition. We have shown previously that DT interacts with planar lipid bilayers at acidic pH to produce voltage-dependent channels7. We report here that this interaction of DT with bilayers depends on membrane phospholipid composition; the interaction is optimal when inositides are present within the membrane, and the inositides are required on the side of the membrane opposite to that in which DT is introduced.

SPECIFIC Escherichia coli DNA fragments carrying flagellar genes have been cloned on λ vehicles and gene expression has been studied in λ-infected ultraviolet irradiated-cells1,2. This work has led to the identification of several gene products involved in flagellar function. But, the structural gene for the hook subunit protein, which is one of the major flagellar components has not been identified. This gene could have been missed if it was not part of the DNA that was cloned, if it was cleaved during endonuclease treatment or if it was transcribed at such a low level that the product could not be detected in ultraviolet-irradiated cells. We report here another approach which avoids these difficulties and has enabled us to identify the region of the genome which carries genes involved in the synthesis of flagellar hook and basal structures. This result is interesting for two reasons. First, it demonstrates the general applicability of cloning techniques to the identification of any gene product in E. coli. Second, it focuses attention on a hitherto poorly defined group of genes involved in flagellar structure and function.

THE specific interaction of many components is necessary for the assembly and function of flagella in Escherichia coli. Twenty genes have been shown to participate1–3 and at least eleven polypeptides are associated with the structure of the flagellar organelle4. The synthesis and assembly of these components and of flagellin, the major flagellar subunit protein, is tightly coupled and regulated5–7. It has not been possible to analyse these regulatory mechanisms in great detail because: (1) the tight coupling between events usually leads either to the synthesis of a complete organelle, or to no detectable flagellar structure, and (2) most of the flagellar components are present at low levels and their synthesis is difficult to measure. To analyse the hierarchy of control and regulation in this system we have prepared a series of hybrid transducing phages that carry different combinations of flagellar genes. We describe here one of these phages and show that it can be used to study the expression of flagellar gene activities.

The transforming genes of acutely transforming retroviruses are derived from conserved cellular genes (c-onc genes) which are believed to be important in normal cell growth and differentiation1–4. Recent studies indicate that altered expression of c-onc genes, for example, by insertion of viral genomes5,6, gene amplification7,8 or chromosomal translocation9–12, can lead to development of malignant diseases in man and animals. c-myb and c-fes are homologues of the transforming genes of avian myeloblastosis virus and feline sarcoma virus (Gardner and Snyder–Theilen strains), respectively. c-myb is transcribed preferentially in immature haematopoietic cells13 and probably codes for a protein important in differentiation of these cells. The viral fes gene, like several other viral onc genes, encodes a tyrosine-specific protein kinase14. However, c-fes transcripts have not been detected in the types of human cells examined so far15,16. c-myb and c-fes have been assigned to human chromosomes 6 and 15, respectively17,18. Specific aberrations involving these chromosomes have been observed at high frequency in several human neoplasms19. We have now sub-localized c-myb to 6q22–24 and c-fes to 15q25–26 by in situ hybridization of the human c-onc probes to human mitotic chromosome preparations20,21. These chromosomal segments are indeed involved in nonrandom translocations in several human tumours. The results encourage further investigation into the role of onc genes in the pathogenesis of specific neoplasms.

THERE has been extensive study of the genetic basis for flagellar formation1,2, motility3 and chemotaxis4,5 in Escherichia coli. But in order to understand the specific mechanisms involved in these processes it is necessary to identify the gene products. We recently showed how E. coli–λ hybrids could be used to identify flagella-related proteins6 and we have subsequently isolated two phages that carry the genes directly involved in flagellar functions. One phage (Fig. 1a,λ fla2) carries flal7 which regulates the synthesis of flagellar proteins and mot, which is necessary for flagellar rotation, λfla2 also seems to carry part of the cheA gene8, which is concerned with regulating flagellar rotation in response to chemotactic signals. It is thought to carry only part of the cheA gene because the phage does not show genetic complementation when it is introduced into cheA mutant strains. It does, however, produce wild-type recombinants with one cheA mutant (e14t1) but not with another (e14q1) (Fig. 1b). Furthermore, the hybrid-λ, phage that carries the adjacent segment of DNA (Fig. 1a, λfla3) also does not show complementation when it is introduced into cheA mutant strains. It gives wild-type recombinants with the cheA mutant e14ql but not with the strain that carries the allele e14tl (Fig. 1b). On the basis of these observations we concluded that the endonuclease (EcoRI) which was used to generate the DNA fragments that were cloned cleaved the cheA gene, and that λfla2 carries the promoter proximal part of the gene8 while λfla3 carries the distal part of the gene (Fig. 1a).

Site-specific recombination reactions involve the joining or rearrangement of discrete DNA segments in a highly precise manner. A site-specific DNA inversion regulates the expression of flagellin genes in Salmonella by switching the orientation of a promoter1,2. Analysis of the reaction has shown that, in addition to DNA sequences at the two boundaries of the 1-kilobase invertible segment where strand exchange occurs, another cis acting sequence is required for efficient inversion3. This 60-base-pair enhancer-like sequence can function at many different locations and in either orientation in a plasmid substrate. It includes two binding sites for a host protein called Factor II or Fis (refs 4 and 5). Here we have investigated the importance of the spatial relationship between the two Fis binding sites for enhancer activity and have found that the correct helical positioning of the binding sites on the DNA is critical. However, this result could not be accounted for by effects on Fis binding. We propose a model for enhancer function in which the enhancer region acts to align the recombination sites into a specific conformation required for productive synapsis.

AMBROSE et al. 1 have shown that individual tumour cells from hamster kidney possess a significantly greater electrophoretic mobility than normal homologous kidney cells. Later, Purdom et al. 2 obtained a correlation between the development of a mouse sarcoma and the progressive increase in the negative electrophoretic mobility of individual cells obtained from this sarcoma at various stages of its development. As the tumour cell acquired more malignant properties, evinced by the formation of early metastases, the negative electrophoretic mobility of individual cells was observed to have increased.

BACTERIAL flagella are generally composed of three morphologically distinguishable regions: (a) the long flagellar filament which accounts for more than 95% of the flagellar protein; (b) the hook, which is generally 80–90 nm long and has a characteristic shape, and (c) the basal structure which is composed of an intricate set of disks and rods attaching the hook to the cell membrane and cell wall1–3.

IT has been recognized that the very high surface brightness temperatures of pulsars at radio frequencies imply that the radiation at these frequencies must be produced by some type of coherent emission mechanism. Chiu and Canuto (ref. 1 and unpublished work) have proposed that such emission is due to a modified bremsstrahlung mechanism which may take place in the very strong-magnetic fields expected to be associated with neutron stars. They have argued that oscillations of the neutron star may cause a population inversion of the radiating electrons, thus possibly giving rise to coherent radiation. The purpose of this communication is to point out that their modified bremsstrahlung mechanism cannot, for any electron distribution, lead to coherent radiation.

IT is well known that the magnetic permeability of ferromagnetic materials decreases towards unity in the region of centimetre waves. The measurements by Hoag and Jones1 extend only down to 20 cm. wave-length (undamped oscillations) ; all the older measurements used damped oscillations.

WE have shown1 that a light curve similar to that of the quasi-stellar source 3C 273 can be generated by a superposition of unit light curves occurring at random times. Gudzenko, Ozernoy and Chertoprud2 have argued (1) that the distribution of the observed luminosities should be expected to be Gaussian on our model, and (2) that the observed distribution is not Gaussian, so that the luminosity curve cannot be regarded as the superposition of random events. The purpose of this communication is to question the validity of both arguments and to show that the luminosity record may still be considered to be of random origin.

IT is generally known that the juvenility of plant organs can be prolonged by treatment with kinetin; moreover, senescent tissues can be rejuvenated1, and several authors have shown increased mutability of older tissues2. Treatment of plant leaves by kinetin appreciably inhibits the multiplication of viruses3,4, although the virus contains RNA. After incorporation into the host cells, the virus may cause information disturbances by inducing the synthesis of foreign RNA. In this interpretation, the changes brought about by the virus are in the nature of mutations.