Striking sequence similarity in inter- and intra-specific comparisons of class I SLG alleles from Brassica oleracea and Brassica campestris: Implications for the evolution and recognition mechanism

Self-incompatibility in Brassica is controlled by a single multi-allelic locus (S locus), which contains at least two highly polymorphic genes expressed in the stigma: an S glycoprotein gene (SLG) and an S receptor kinase gene (SRK). The putative ligand-binding domain of SRK exhibits high homology to the secretory protein SLG, and it is believed that SLG and SRK form an active receptor kinase complex with a self-pollen ligand, which leads to the rejection of self-pollen. Here, we report 31 novel SLG sequences of Brassica oleracea and Brassica campestris. Sequence comparisons of a large number of SLG alleles and SLG-related genes revealed the following points. (i) The striking sequence similarity observed in an inter-specific comparison (95.6% identity between SLG14 of B. oleracea and SLG25 of B. campestris in deduced amino acid sequence) suggests that SLG diversification predates speciation. (ii) A perfect match of the sequences in hypervariable regions, which are thought to determine S specificity in an intra-specific comparison (SLG8 and SLG46 of B. campestris) and the observation that the hypervariable regions of SLG and SRK of the same S haplotype were not necessarily highly similar suggests that SLG and SRK bind different sites of the pollen ligand and that they together determine S specificity. (iii) Comparison of the hypervariable regions of SLG alleles suggests that intragenic recombination, together with point mutations, has contributed to the generation of the high level of sequence variation in SLG alleles. Models for the evolution of SLG/SRK are presented.

Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus

We report the crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus, a major human pathogen, to 2.8-Å resolution. This enzyme is a key target for developing specific antiviral therapy. The structure of the catalytic domain contains 531 residues folded in the characteristic fingers, palm, and thumb subdomains. The fingers subdomain contains a region, the “fingertips,” that shares the same fold with reverse transcriptases. Superposition to the available structures of the latter shows that residues from the palm and fingertips are structurally equivalent. In addition, it shows that the hepatitis C virus polymerase was crystallized in a closed fingers conformation, similar to HIV-1 reverse transcriptase in ternary complex with DNA and dTTP [Huang H., Chopra, R., Verdine, G. L. & Harrison, S. C. (1998) Science 282, 1669–1675]. This superposition reveals the majority of the amino acid residues of the hepatitis C virus enzyme that are likely to be implicated in binding to the replicating RNA molecule and to the incoming NTP. It also suggests a rearrangement of the thumb domain as well as a possible concerted movement of thumb and fingertips during translocation of the RNA template-primer in successive polymerization rounds.

Heme is an effector molecule for iron-dependent degradation of the bacterial iron response regulator (Irr) protein

The bacterial iron response regulator (Irr) protein mediates iron-dependent regulation of heme biosynthesis. Pulse–chase and immunoprecipitation experiments showed that Irr degraded in response to 6 μM iron with a half-life of ≈30 min and that this regulated stability was the principal determinant of control by iron. Irr contains a heme regulatory motif (HRM) near its amino terminus. A role for heme in regulation was implicated by the retention of Irr in heme synthesis mutants in the presence of iron. Addition of heme to low iron (0.3 μM) cultures was sufficient for the disappearance of Irr in cells of the wild-type and heme mutant strains. Spectral and binding analyses of purified recombinant Irr showed that the protein bound heme with high affinity and caused a blue shift in the absorption spectrum of heme to a shorter wavelength. A Cys29 → Ala substitution within the HRM of Irr (IrrC29A) abrogated both high affinity binding to heme and the spectral blue shift. In vivo turnover experiments showed that, unlike wild-type Irr, IrrC29A was stable in the presence of iron. We conclude that iron-dependent degradation of Irr involves direct binding of heme to the protein at the HRM. The findings implicate a regulatory role for heme in protein degradation and provide direct evidence for a functional HRM in a prokaryote.

Oncogenic stimulation recruits cyclin-dependent kinase in the cell cycle start in rat fibroblast

The rat fibroblast NRK cells are transformed reversibly by a combination of growth factors. When stimulated with serum, NRK cells rely on cyclin-dependent kinase 4 (Cdk4) for their S phase entry. However, when stimulated with serum containing oncogenic growth factors, they come to rely on either Cdk4 or Cdk6, and their S phase entry cannot be blocked unless both Cdk4 and Cdk6 are immunodepleted. Such change of dependence does not occur in the NRK cell mutants defective in an oncogenic signal pathway and, therefore, deficient in anchorage-independent cell cycle start ability, correlating Cdk6 dependence with this remarkable, cancer-associated phenotype. However, both Cdk4 and Cdk6 are activated upon serum stimulation, and neither the amounts of Cdk6, Cdk4, cyclin D1, and cyclin-dependent kinase inhibitors nor the activities or subcellular localization of Cdk6 and Cdk4 are significantly influenced by oncogenic stimulation. Thus, oncogenic stimulation invokes Cdk6 to participate in a critical step of the cell cycle start in a rat fibroblast, but by a mechanism seemingly unrelated to the regulation of the kinase. Given that many hematopoietic cells employ predominantly Cdk6 for the cell cycle start and perform anchorage-independent growth by nature, our results raise the possibility that the oncogenic stimulation-induced anchorage-independent cell cycle start of NRK is elicited by a mechanism similar to the one used for hematopoietic cell proliferation.

Postnatal neuronal proliferation in mice lacking Ink4d and Kip1 inhibitors of cyclin-dependent kinases

Development of the central nervous system requires proliferation of neuronal and glial cell precursors followed by their subsequent differentiation in a highly coordinated manner. The timing of neuronal cell cycle exit and differentiation is likely to be regulated in part by inhibitors of cyclin-dependent kinases. Overlapping and sustained patterns of expression of two cyclin-dependent kinases, p19Ink4d and p27Kip1, in postmitotic brain cells suggested that these proteins may be important in actively repressing neuronal proliferation. Animals derived from crosses of Ink4d- null with Kip1-null mice exhibited bradykinesia, proprioceptive abnormalities, and seizures, and died at about 18 days after birth. Metabolic labeling of live animals with bromodeoxyuridine at postnatal days 14 and 18, combined with immunolabeling of neuronal markers, showed that subpopulations of central nervous system neurons were proliferating in all parts of the brain, including normally dormant cells of the hippocampus, cortex, hypothalamus, pons, and brainstem. These cells also expressed phosphorylated histone H3, a marker for late G2 and M-phase progression, indicating that neurons were dividing after they had migrated to their final positions in the brain. Increased proliferation was balanced by cell death, resulting in no gross changes in the cytoarchitecture of the brains of these mice. Therefore, p19Ink4d and p27Kip1 cooperate to maintain differentiated neurons in a quiescent state that is potentially reversible.

Novel dimeric interface and electrostatic recognition in bacterial Cu,Zn superoxide dismutase

Eukaryotic Cu,Zn superoxide dismutases (CuZnSODs) are antioxidant enzymes remarkable for their unusually stable β-barrel fold and dimer assembly, diffusion-limited catalysis, and electrostatic guidance of their free radical substrate. Point mutations of CuZnSOD cause the fatal human neurodegenerative disease amyotrophic lateral sclerosis. We determined and analyzed the first crystallographic structure (to our knowledge) for CuZnSOD from a prokaryote, Photobacterium leiognathi, a luminescent symbiont of Leiognathid fish. This structure, exemplifying prokaryotic CuZnSODs, shares the active-site ligand geometry and the topology of the Greek key β-barrel common to the eukaryotic CuZnSODs. However, the β-barrel elements recruited to form the dimer interface, the strategy used to forge the channel for electrostatic recognition of superoxide radical, and the connectivity of the intrasubunit disulfide bond in P. leiognathi CuZnSOD are discrete and strikingly dissimilar from those highly conserved in eukaryotic CuZnSODs. This new CuZnSOD structure broadens our understanding of structural features necessary and sufficient for CuZnSOD activity, highlights a hitherto unrecognized adaptability of the Greek key β-barrel building block in evolution, and reveals that prokaryotic and eukaryotic enzymes diverged from one primordial CuZnSOD and then converged to distinct dimeric enzymes with electrostatic substrate guidance.

Glutamate transport in Rhodobacter sphaeroides is mediated by a novel binding protein-dependent secondary transport system

Growth of a glutamate transport-deficient mutant of Rhodobacter sphaeroides on glutamate as sole carbon and nitrogen source can be restored by the addition of millimolar amounts of Na+. Uptake of glutamate (Kt of 0.2 μM) by the mutant strictly requires Na+ (Km of 25 mM) and is inhibited by ionophores that collapse the proton motive force (pmf). The activity is osmotic-shock-sensitive and can be restored in spheroplasts by the addition of osmotic shock fluid. Transport of glutamate is also observed in membrane vesicles when Na+, a proton motive force, and purified glutamate binding protein are present. Both transport and binding is highly specific for glutamate. The Na+-dependent glutamate transporter of Rb. sphaeroides is an example of a secondary transport system that requires a periplasmic binding protein and may define a new family of bacterial transport proteins.

A zinc finger directory for high-affinity DNA recognition

We have used two monovalent phage display libraries containing variants of the Zif268 DNA-binding domain to obtain families of zinc fingers that bind to alterations in the last 4 bp of the DNA sequence of the Zif268 consensus operator, GCG TGGGCG. Affinity selection was performed by altering the Zif268 operator three base pairs at a time, and simultaneously selecting for sets of 16 related DNA sequences. In this way, only four experiments were required to select for all possible 64 combinations of DNA triplet sequences. The results show that (i) for high-affinity DNA binding in the range observed for the Zif268 wild-type complex (Kd = 0.5–5 nM), finger 1 specifically requires the arginine at the carboxy terminus of its recognition helix that forms a bidentate hydrogen-bond with the guanine base (G) in the crystal structure of Zif268 complexed to its DNA operator sequence GCG TGG GCG; (ii) when the guanine base (G) is replaced by A, C, or T, a lower-affinity family (Kd ⩾ 50 nM) can be detected that shows an overall tendency to bind G-rich DNA; (iii) the residues at position 2 on the finger 2 recognition helix do not appear to interact strongly with the complementary 5′ base in the finger 1 binding site; and (iv) unexpected substitutions at the amino terminus of finger 1 can occasionally result in specificity for the 3′ base in the finger 1 binding site. A DNA recognition directory was constructed for high-affinity zinc fingers that recognize all three bases in a DNA triplet for seven sequences of the type GNN. Similar approaches may be applied to other zinc fingers to broaden the scope of the directory.

A novel RNA polymerase I-dependent RNase activity that shortens nascent transcripts from the 3′ end

A novel RNase activity was identified in a yeast RNA polymerase I (pol I) in vitro transcription system. Transcript cleavage occurred at the 3′ end and was dependent on the presence of ternary pol I/DNA/RNA complexes and an additional protein factor not identical to transcription factor IIS (TFIIS). Transcript cleavage was observed both on arrested complexes at the linearized ends of the transcribed DNA and on intrinsic blocks of the DNA template. Shortened transcripts that remained associated within the ternary complexes were capable of resuming RNA chain elongation. Possible functions of the nuclease for transcript elongation or termination are discussed.

Molecular basis for the recognition of two structurally different major histocompatibility complex/peptide complexes by a single T-cell receptor

2C is a typical alloreactive cytotoxic T lymphocyte clone that recognizes two different ligands. These ligands are adducts of the allo-major histocompatibility complex (MHC) molecule H-2Ld and an endogenous octapeptide, and of the self-MHC molecule H-2Kb and another peptide. MHC-binding and T-cell assays with synthetic peptides in combination with molecular modeling studies were employed to analyze the structural basis for this crossreactivity. The molecular surfaces of the two complexes differ greatly in densities and distributions of positive and negative charges. However, modifications of the peptides that increase similarity decrease the capacities of the resulting MHC peptide complexes to induce T-cell responses. Moreover, the roles of the peptides in ligand recognition are different for self- and allo-MHC-restricted T-cell responses. The self-MHC-restricted T-cell responses were finely tuned to recognition of the peptide. The allo-MHC-restricted responses, on the other hand, largely ignore modifications of the peptide. The results strongly suggest that adaptation of the T-cell receptor to the different ligand structures, rather than molecular mimicry by the ligands, is the basis for the crossreactivity of 2C. This conclusion has important implications for T-cell immunology and for the understanding of immunological disorders.

γ-Aminobutyric acid type B receptor-dependent burst-firing in thalamic neurons: A dynamic clamp study

Synchronized network responses in thalamus depend on phasic inhibition originating in the thalamic reticular nucleus (nRt) and are mediated by the neurotransmitter γ-aminobutyric acid (GABA). A suggested role for intra-nRt connectivity in inhibitory phasing remains controversial. Recently, functional GABA type B (GABAB) receptors were demonstrated on nRt cells, and the slow time course of the GABAB synaptic response seems ideally suited to deinactivate low-threshold calcium channels. This promotes burst firing, a characteristic feature of synchronized responses. Here we investigate GABAB-mediated rebound burst firing in thalamic cells. Whole-cell current-clamp recordings were obtained from nRt cells and somatosensory thalamocortical relay cells in rat brain slices. Synthetic GABAB inhibitory postsynaptic potentials, generated by a hybrid computer–neuron synapse (dynamic clamp), triggered rebound low-threshold calcium spikes in both cell types when peak inhibitory postsynaptic potential hyperpolarization was greater than −92 mV. The threshold inhibitory postsynaptic potential conductance for rebound burst generation was comparable in nRt (7 nS) and thalamocortical (5 nS) cells. However, burst onset in nRt (1 s) was considerably delayed compared with thalamocortical (0.6 s) cells. Thus, GABAB inhibitory postsynaptic potentials can elicit low-threshold calcium spikes in both relay and nRt neurons, but the resultant oscillation frequency would be faster for thalamocortical–nRt networks (3 Hz) than for nRt–nRt networks (1–2 Hz). We conclude, therefore, that fast (>2 Hz) GABAB-dependent thalamic oscillations are maintained primarily by reciprocal connections between excitatory and inhibitory cells. These findings further indicate that when oscillatory neural networks contain both recurrent and reciprocal inhibition, then distinct population frequencies may result when one or the other type of inhibition is favored.

Calcium-dependent switching of the specificity of phosphoinositide binding to synaptotagmin

The synaptic vesicle membrane protein synaptotagmin (tagmin) is essential for fast, calcium-dependent, neurotransmitter release and is likely to be the calcium sensor for exocytosis, because of its many calcium-dependent properties. Polyphosphoinositides are needed for exocytosis, but it has not been known why. We now provide a possible connection between these observations with the finding that the C2B domain of tagmin I binds phosphatidylinositol-4,5-bisphosphate (PIns-4,5-P2), its isomer phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate (PIns-3,4,5-P3). Calcium ions switch the specificity of this binding from PIns-3,4,5-P3 (at calcium concentrations found in resting nerve terminals) to PIns-4,5-P2 (at concentration of calcium required for transmitter release). Inositol polyphosphates, known blockers of neurotransmitter release, inhibit the binding of both PIns-4,5-P2 and PIns-3,4,5-P3 to tagmin. Our findings imply that tagmin may operate as a bimodal calcium sensor, switching bound lipids during exocytosis. This connection to polyphosphoinositides, compounds whose levels are physiologically regulated, could be important for long-term memory and learning.

Ca2+-induced rebound potentiation of γ-aminobutyric acid-mediated currents requires activation of Ca2+/calmodulin-dependent kinase II

In cerebellar Purkinje neurons, γ-aminobutyric acid (GABA)-mediated inhibitory synaptic transmission undergoes a long-lasting “rebound potentiation” after the activation of excitatory climbing fiber inputs. Rebound potentiation is triggered by the climbing-fiber-induced transient elevation of intracellular Ca2+ concentration and is expressed as a long-lasting increase of postsynaptic GABAA receptor sensitivity. Herein we show that inhibitors of the Ca2+/calmodulin-dependent protein kinase II (CaM-KII) signal transduction pathway effectively block the induction of rebound potentiation. These inhibitors have no effect on the once established rebound potentiation, on voltage-gated Ca2+ channel currents, or on the basal inhibitory transmission itself. Futhermore, a protein phosphatase inhibitor and the intracellularly applied CaM-KII markedly enhanced GABA-mediated currents in Purkinje neurons. Our results demonstrate that CaM-KII activation and the following phosphorylation are key steps for rebound potentiation.

Is redshift-dependent evolution of galaxies a theoretical artifact?

The physical validity of the hypothesis of (redshift-dependent) luminosity evolution in galaxies is tested by statistical analysis of an intensively studied complete high-redshift sample of normal galaxies. The necessity of the evolution hypothesis in the frame of big-bang cosmology is confirmed at a high level of statistical significance; however, this evolution is quantitatively just as predicted by chronometric cosmology, in which there is no such evolution. Since there is no direct observational means to establish the evolution postulated in big-bang studies of higher-redshift galaxies, and the chronometric predictions involve no adjustable parameters (in contrast to the two in big-bang cosmology), the hypothesized evolution appears from the standpoint of conservative scientific methodology as a possible theoretical artifact.

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA′-′lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.