To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We demonstrate the allotetraploid origin of X. laevis by partitioning its genome into two homeologous subgenomes, marked by distinct families of “fossil” transposable elements. Based on the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged ~34 million years ago (Mya) and combined to form an allotetraploid ~17–18 Mya. 56% of all genes are retained in two homeologous copies. Protein function, gene expression, and the amount of flanking conserved sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.

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hairy is a Drosophila pair-rule segmentation gene that functions genetically as a repressor. To isolate protein components of Hairy-mediated repression, we used a yeast interaction screen and identified a Hairyinteracting protein, the Drosophila homolog of the human C-terminal-binding protein (CtBP). Human CtBP is a cellular phosphoprotein that interacts with the C-terminus of the adenovirus E1a oncoprotein and functions as a tumor suppressor. dCtBP also interacts with E1a in a directed yeast two-hybrid assay. We show that dCtBP interacts specifically and directly with a small, previously uncharacterized C-terminal region of Hairy. dCtBP activity appears to be specific to Hairy of the Hairy/Enhancer of split [E(spl)]/Dpn basic helix-loop-helix protein class. We identified a Pelement insertion within the dCtBP transcription unit that fails to complement alleles of a known locus, l(3)87De. We demonstrate that dCtBP is essential for proper embryonic segmentation by analyzing embryos lacking maternal dCtBP activity. While Hairy is probably not the only segmentation gene interacting with dCtBP, we show dose-sensitive genetic interactions between dCtBP and hairy mutations.

The left-right (L-R) asymmetric expression of lefty2 and nodal is controlled by a left side-specific enhancer (ASE). The transcription factor FAST2, which can mediate signaling by TGF beta and activin, has now been identified as a protein that binds to a conserved sequence in ASE. These FAST2 binding sites were both essential and sufficient for L-R asymmetric gene expression. The Fast2 gene is bilaterally expressed when nodal and lefty2 are expressed on the left side. TGF beta and activin can activate the ASE activity in a FAST2-dependent manner, while Nodal can do so in the presence of an EGF-CFC protein. These results suggest that the asymmetric expression of lefty2 and nodal is induced by a left side-specific TGF beta-related factor, which is most likely Nodal itself.

Two types of polycations with pendant active groups were synthesized: one is polymethacrylate containing pendant biguanide units, and the other is poly(vinylbenzyl ammonium chloride). The two polycations were found to exhibit higher bactericidal activity against Staphylococcus aureus than the corresponding monomers.Fractionation of the polycations was successfully performed on gel filtration chromatography, and examination of the antibacterial activity against S. aureus of the well-characterized polymer samples with various molecular weights (MW) revealed that the activity was strongly dependent on the MW of the polycations and that there existed an optimal MW range for the cidal action of the polymeric biocides. Experiments on the lysis of protoplasts ofBacillus subtilis in contact with the polycations have shown that the target sites of the polycationic biocides are cytoplasmic membranes of bacteria.In previous papers, we reported that in a clean system where there are no interfering materials such as negatively charged macromolecules, polycations having biguanide units or quaternary ammonium salts exhibit much higher antibacterial activity than the monomeric analogs (4, 5). In view of the fact that many properties of polymers depend strongly on their molecular weights (MW) and MW distributions, it is reasonable to expect that the antibacterial activity of such polycations is MW dependent. In fact, in the oligomers of in-chain quaternary ammonium salts ranging from monomer to tetramer, a strong MW dependence has been observed (T. Ikeda, H. Yamaguchi, and S. Tazuke, submitted for publication).In this paper, we describe the preparation of wellcharacterized polycationic samples with various MW and narrow MW distributions. In addition, we report on the effect of MW of the polycations on bactericidal activity, focusing our attention on the high-MW polymers. Various physiological events were observed in intact cells and protoplasts when they were exposed to various polycations. The effects are discussed in terms of the mode of action of these polycationic biocides. MATERIALS AND METHODSPreparation. The synthetic route for the methacrylate monomer with a pendant biguanide is shown in Fig. 1. 2-Hydroxyethylphenyldicyandiamide (compound 1) was prepared as reported previously (5). To prepare 4-(2-methacryloyloxyethyl)phenyldicyandiamide (compound 2), a solution of compound 1 (14 g, 0.07 mol) dissolved in tetrahydrofuran-water (10:1, 93 ml) was cooled in an ice bath; to this solution, freshly distilled methacryloyl chloride (52 ml, 0.54 mol) was added dropwise with stirring over a period of 2 h; and the reaction mixture was left overnight at room temperature. It was then poured into a large excess of water, and the precipitate was collected and dried under vacuum (yield, 90%). The product was recrystallized from 2-propanol. mp 181-182°C; NMR (dimethyl sulfoxide-d6, 8)

Electrical events and intracellular calcium concentration ([Ca2+]) imaged using fluo‐3 and laser scanning confocal microscopy were simultaneously monitored in single smooth muscle cells freshly isolated from guinea‐pig vas deferens or urinary bladder. Images obtained every 8 ms, during stepping from ‐60 to 0 or +10 mV for 50 ms under voltage clamp, showed that a rise in [Ca2+] could be detected within 20 ms of depolarization in five to twenty small (< 2 μm diameter) ‘hot spots’, over 95 % of which were located within 1.5 μm of the cell membrane. Depolarization at 30 s intervals activated hot spots at the same places. Cd2+ or verapamil abolished both hot spots and Ca2+‐activated K+ current (IK,Ca). Caffeine almost abolished hot spots and markedly reduced IK,Ca. Cyclopiazonic acid, which raised basal global [Ca2+], decreased the rise in hot spot [Ca2+] and IK,Ca amplitude during depolarization. These results suggest that Ca2+ entry caused Ca2+‐induced Ca2+ release (CICR). Under voltage clamp, hot spot [Ca2+] closely paralleled the rise in IK,Ca and reached a peak within 20 ms of the start of depolarization, but the rise in global [Ca2+] over the whole cell area was much slower. Step depolarization to potentials positive to ‐20 mV caused hot spots to grow in size and coalesce, leading to a rise in global [Ca2+] and contraction. Ca2+ hot spots also occurred during the up‐stroke of an evoked action potential under current clamp. It is concluded that the entry of Ca2+ in the early stages of an action potential evokes CICR from discrete subplasmalemma Ca2+ storage sites and generates hot spots that spread to initiate a contraction. The activation of Ca2+‐dependent K+ channels in the plasmalemma over hot spots initiates IK,Ca and action potential repolarization.

SUMMARY1. Ionic currents underlying the action potential were recorded from enzymatically isolated smooth muscle cells of guinea-pig ureter.2. The action potential recorded from a single cell was similar to that from a multicellular preparation. It showed repetitive spikes on a plateau potential which followed the first spike. Treatment with 10 mM-tetraethylammonium (TEA) increased the amplitude and duration of the plateau phase and abolished the repetitive spikes.3. Under voltage clamp mode, at least two (maybe three) kinds of outward currents were activated during depolarizing pulses. The main outward current was Ca2"-dependent K+ current (IK(ca)), which was mostly blocked in Ca2`-free solution, or by application of 1 mM-cadmium (Cd2+) or 2 mM-tetraethylammonium (TEA).IK(ca) was greatly decreased by treatment with 5 mM-caffeine or an addition of 10 mM-EGTA in a pipette solution.4. In the presence of 1 mM-Cd2' and 2 mM-TEA, a small transient outward current remained. 4-Aminopyridine (1 mM) suppressed the transient outward current by about 40 %. Time-and voltage-dependent delayed rectifier outward currents were small in ureter cells. An inwardly rectifying K+ current was not detected.5. An application of 1 mM-Cd2+, 5 mM-cobalt (Co2+), 1 mM-lanthanum (La3+) or 0-1 IuM-nifedipine completely blocked the action potential. Replacement of 80-90 % of extracellular Na+ with Li+ or Tris almost abolished the plateau potential and repetitive spikes but did not change significantly the first spike. 6. In the presence of 30 mM-TEA, the inward current elicited by depolarization was monophasic and lasted for more than 1 s. Application of 1 mM-Cd2+, 1 mM-La3 , 0-1 /LM-nifedipine, or 5 mM-Co2+ completely blocked inward current. The replacement (87 %) of extracellular Na+ ions with Li+, Tris, sucrose or TEA speeded up the decay of inward current; the inward current decreased by 10-60 % at the end of a 500 ms pulse.7. Even in low-Na+ solution (120 mM-TEA), the inactivation of ICa had a quite slow component (T = 1 S), in addition to another faster component (T = 100 ms) at 0 mV. When short depolarizing clamp pulses (50 ms) were repetitively applied at short intervals (50 ms) and with interpulse voltage of -10 or -20 mV to mimic the repetitive spikes on the plateau of the action potential, the decline of peak Ca2+ 5-2 Y. IMAIZUMI, K. MURAKI AND M. WATANABE current during the train of pulses was smaller than the decay of Ca2+ current during a long pulse.8. These results indicate that (1) the repetitive spikes on the plateau phase of the action potential are attributable to the slow inactivation and rapid reactivation of I4a and repetitive activation of IK(Ca) due to Ca2+-induced Ca2+ release from stores, and (2) the long plateau phase in the action potential may be due to a slowly inactivating Iba, a Na+-dependent late inward current, and a small delayed rectifier outward current.

The relationship between Ca2+ sparks spontaneously occurring at rest and local Ca2+ transients elicited by depolarization was analysed using two‐dimensional confocal Ca2+ images of single smooth muscle cells isolated from guinea‐pig vas deferens and urinary bladder. The current activation by these Ca2+ events was also recorded simultaneously under whole‐cell voltage clamp. Spontaneous transient outward currents (STOCs) and Ca2+ sparks were simultaneously detected at ‐40 mV in approximately 50 % of myocytes of either type. Ca2+ sparks and corresponding STOCs occurred repetitively in several discrete sites in the subplasmalemmal area. Large conductance Ca2+‐dependent K+ (BK) channel density in the plasmalemma near the Ca2+ spark sites generating STOCs was calculated to be 21 channels μm−2. When myocytes were depolarized from ‐60 to 0 mV, several local Ca2+ transients were elicited within 20 ms in exactly the same peripheral sites where sparks occurred at rest. The local Ca2+ transients often lasted over 300 ms and spread into other areas. The appearance of local Ca2+ transients occurred synchronously with the activation of Ca2+‐dependent K+ current (IK,Ca). Immunofluorescence staining of the BK channel α‐subunit (BKα) revealed a spot‐like pattern on the plasmalemma, in contrast to the uniform staining of voltage‐dependent Ca2+ channel α1C subunits along the plasmalemma. Ryanodine receptor (RyR) immunostaining also suggested punctate localization predominantly in the periphery. Double staining of BKα and RyRs revealed spot‐like co‐localization on/beneath the plasmalemma. Using pipettes of relatively low resistance, inside‐out patches that included both clustered BK channels at a density of over 20 channels μm−2 and functional Ca2+ storage sites were obtained at a low probability of ≈5 %. The averaged BK channel density was 3‐4 channels μm−2 in both types of myocyte. These results support the idea that a limited number of discrete sarcoplasmic reticulum (SR) fragments in the subplasmalemmal area play key roles in the control of BK channel activity in two ways: (i) by generating Ca2+ sparks at rest to activate STOCs and (ii) by generating Ca2+ transients presumably triggered by sparks during an action potential to activate a large IK,Ca and also induce a contraction. BK channels and RyRs may co‐localize densely at the junctional areas of plasmalemma and SR fragments, where Ca2+ sparks occur to elicit STOCs.