Macromolecular structure of nuclear polyhedrosis virus genome

Summary DNA preparations from nuclear polyhedrosis virus (NPV) ofGalleria mellonella L. (GmL) were fractionated in high ionic strength neutral sucrose gradient. This procedure allowed a separation of supercoiled infectious DNA molecules with contour length of 48–52 µ from infectious open ring DNA molecule, and non-infectious linear DNA molecules of the same size. In addition a heterogeneity of supercoiled DNA molecules was detected. Covalently closed DNA molecules did not contain protein or ribonucleotide ligands which could be digested by pronase or pancreatic RNase treatment.It is concluded from data on the infectivity of different molecular forms of DNA and reassociation kinetics studies, that the genome of GmL NPV is a unique ring nucleotide sequence with a molecular weight of about 90–100×10⁶.With 8 Figures     

Helicase Hmi1 stimulates the synthesis of concatemeric mitochondrial DNA molecules in yeast Saccharomyces cerevisiae

Hmi1p is a helicase in the yeast Saccharomyces cerevisiae required for maintenance of the wild-type mitochondrial genome. Disruption of the HMI1 ORF generates ^– and ⁰ cells. Here we demonstrate that, in ^– yeast strains, Hmi1p stimulates the synthesis of long concatemeric mitochondrial DNA molecules associated with a reduction in the number of nucleoids used for mitochondrial DNA packaging. Surprisingly, the ATPase negative mutants of Hmi1p can also stimulate the synthesis of long concatemeric ^– mitochondrial DNA molecules and support the maintenance of the wild-type mitochondrial genome, albeit with reduced efficiency. We show that, in the mutant hmi1–5 background, the wild-type mitochondrial DNA is fragmented; and we propose that, in hmi1 yeast cells, the loss of the wild-type mitochondrial genome is caused by this fragmentation of the mitochondrial DNA.     

Biochemical and genetic characterization of vaccinia virus temperature-sensitive mutants with DNA− and DNAf− phenotypes

Summary Eighty temperature-sensitive(ts) mutants of vaccinia virus were examined for defects in synthesis of DNA. Ninets mutants were incapable of synthesizing DNA at the restrective temperature of 39.5° C (DNA^– mutants). Biochemical and genetic data indicate that all 9 DNA^– mutants carry mutations in different genes. Temperature shift-up experiments have shown that 6ts mutants with the DNA^– phenotype have mutations in the genes coding for the proteins directly associated with vaccinia DNA synthesis.Temperature shift-down experiments in the presence of cytosine arabinoside revealed 5ts mutants capable of synthesizing DNA at the elevated temperature, but this DNA failed to form infectious virions. Thesets mutants were designated as DNAf^– mutants. Pulse-chase experiments for the DNAf^– mutant 1877 revealed that viral DNA produced at 39.5° C was incapable of entering into mature virions or any subviral particles.Based on the data for recombination amongts mutants with the DNA^– and DNAf^– phenotype a tentative genetic map was constructed.With 3 Figures     

Electron microscopic investigation of mitochondrial DNA fromChenopodium album (L.)

DNA molecules from mitochondria of whole plants and a suspension culture ofChenopodium album were prepared, by a gentle method, for analysis by electron microscopy. Mitochondrial (mt) DNA preparations from both sources contained mostly linear molecules of variable sizes (with the majority of molecules ranging from 40 to 160 kb). Open circular molecules with contour lengths corresponding to 0.3–183 kb represented 23–26% of all mtDNA molecules in the preparations from the suspension culture and 13–15% in the preparations from whole plants. More than 90% of the circular DNA was smaller than 30 kb. Virtually no size classes of the mtDNA molecules could be identified, and circular or linear molecules of the genome size (about 270 kb) were not observed. In contrast, plastid (pt) DNA preparations from the suspension culture contained linear and circular molecules falling into size classes corresponding to monomers, dimers and trimers of the chromosome. About 23% of the ptDNA molecules were circular. DNA preparations from mitochondria contained a higher percentage of more complex molecules (rosette-like structures, catenate-like molecules) than preparations of ptDNA. Sigma-like molecules (putative intermediates of rollingcircle replication) were observed in mtDNA preparations from the suspension culture (18% of the circles), and in much lower amount (1%) in preparations from whole plants. The results are compared with data obtained previously by pulsed-field gel electrophoresis and discussed in relation to the structural organization and replication of the mt genome of higher plants.     

Identification and mapping of origins of DNA replication within the DNA sequences of the genome of insect iridescent virus type 6

The origins of DNA replication of the genome (209 kbp) of Chilo iridescent virus (CIV), which is circularly permuted and terminally redundant, were identified. The defined genomic library of CIV, which represents 100% of DNA sequences of the viral genome (e.g., all 32EcoRI CIV DNA fragments), was used for transfection ofChoristoneura fumiferana insect cell cultures (CF-124) that were previously infected with CIV. The plasmid rescue experiments were carried out to select those recombinant plasmids that were amplified during viral replication in CIV-infected cell cultures. It was found that six recombinant plasmids harboring theEcoRI DNA fragments C [13.5 kbp, 0.909-0.974 map units (m.u.)], H (9.8 kbp, 0.535–0.582 m.u.), M (7.25 kbp, 0.310–0.345 m.u.), O (6.5 kbp, 0.196–0.228 m.u.), Q (5.9 kbp, 0.603–0.631 m.u.), and Y (2.0 kbp, 0.381–0.391 m.u.) were able to be amplified under the conditions used. This indicates that the CIV genome possesses six DNA replication origins. Subclones of theEcoRI CIV DNA fragments C and H were screened under the same conditions. It was found that DNA sequences within theEcoRI DNA fragments C and H at the genome coordinates 0.924–0.930 and 0.535–0.548, respectively, contain origins of viral DNA replication. The DNA nucleotide sequences of theEcoRI CIV DNA fragment Y (1986 bp) were determined for identifying the DNA sequence of the corresponding origin of DNA replication. The computer-aided analysis revealed the presence of a 15-mer inverted repeat at nucleotide positions 661–675 and 677–691 (661-TAAATTTAATGAGAA-G-TTCTCATTAAATTTA-692). The analysis of the DNA sequence of theEcoRI DNA fragment H corresponding to the particular region at the genome coordinates 0.535–0.548 (1) showed that this region contains a 16-mer inverted repeat at the nucleotide positions 1315 and 1332 (1315-TAAATTTTAATGGTTA-A-TAACCATTAAAATTTA-1347), which is very similar to the inverted repetition found within theEcoRI DNA fragment Y. The successful recognition and amplification of the single-stranded synthetic DNA sequences of both strands of CIV-ori-Y (nucleotide position 661–691) using phage M13 system in CIV-infected cells is strong evidence that the CIV-ori-Y is bidirectionally active, and this DNA sequence is considered to be the origin of DNA replication within theEcoRI CIV DNA fragment Y.     

Gene delivery via DNA incorporation within a biomimetic apatite coating

Integrating inductivity with conductivity in a material may advance tissue engineering. An organic/inorganic hybrid was developed by incorporating plasmid DNA encoding for the β-gal gene complexed with Lipofectamine 2000^® (DNA–Lipoplex) within apatite via coprecipitation. It was hypothesized that this system will result in enhanced transfection efficiency compared to DNA–Lipoplexes adsorbed to the mineral surface and DNA coprecipitated without Lipofectamine 2000^®. PLGA films were cast onto glass slips and apatite and DNA were coprecipitated in modified simulated body fluid (mSBF). DNA–Lipoplex presence in mineral, DNA–Lipoplex stability (vs. coprecipitation time), and transfection efficiency (determined with C3H10T1/2 cells) as a function of coprecipitation time, DNA–Lipoplex concentration, and DNA incorporation method were studied. DNA–Lipoplex presence and spatial distribution on apatite were confirmed through fluorescence. Transfection efficiency was highest for 6 h of DNA–Lipoplex coprecipitation. Differences in transfection efficiency were found between the DNA concentrations, with the highest efficiency for coprecipitation being 40 μg/ml (p ≤ 0.009 relative to other coprecipitation concentrations). Significant differences in transfection efficiency existed between incorporation methods (p < 0.05) with the highest efficiency for DNA–Lipoplex coprecipitation. This hybrid material system not only integrates inductivity provided by the DNA and conductivity provided by the apatite, but it also has significant implications in non-viral gene delivery due to its ability to increase transfection efficiency.     

Organization of the 5.8S, 16–18S,and 23–28S ribosomal RNA genes of Cephalosporium acremonium

Summary A 9.2 kb Pst1 restriction fragment, repeated tandemly head-to-tail in the genome, contains the 5.8S, 16–18S, and 23–28S ribosomal RNA (rRNA) genes of Cephalosporium acremonium, a filamentous fungus used at the industrial scale for production of cephalosporin antibiotics. These rRNA genes were located in Pst1 digests of C. acremonium genomic DNA using a hybridization probe that contained the 5.8S, 18S, and 25S rRNA genes from the yeast Saccharomyces cerevisiae. This probe was also used in screening a recombinant lambda library to identify phage carrying rRNA genes of C. acremonium. Yeast rRNA genes contained separately on individual ³²P-labeled plasmids were used to demonstrate that a cloned 7.2 kb C. acremonium sequence, represented in the repeated 9.2 kb Pst1 fragment, contained DNA from the C. acremonium 5.8S, 16–18S, and 23–28S rRNA genes. These genes were ordered with the 5.8S gene located between the 16–18S and 23–28S rRNA genes. The order of the 16–18S, 5.8S, and 23–28S rRNA genes observed in C. acremonium is the same as that observed in rRNA repeats of many other lower eucaryotes, e.g. S. cerevisiae, Aspergillus nidulans, and Neurospora crassa.     

Repair of 2 um Plasmid DNA in Saccharomyces cerevisiae

Summary We have developed a system for assaying pyrimidine dimers in the 2 m DNA plasmid of Saccharomyces cerevisiae, using Micrococcus luteus UV endonuclease to nick dimer-containing plasmid molecules and measuring percentages of nicked and covalently closed circles on agarose gels. UV-irradiation induced dimers in plasmid DNA, in vivo, at the same rate as in chromosomal DNA. After a dose of 20 Joules·m^–2, approximately 86% of plasmid molecules had. at least one dimer. After 3 h incubation under normal growth conditions only 4% still retained dimers in a wild-type strain. In a rad1 (excision-defective) mutant 81% of plasmid molecules still had dimers after 3 h, suggesting that excision repair operates to remove dimers from plasmid DNA in wild-type yeast. Dimers can be removed from 2 ,um DNA in a rad1 mutant by photoreactivation.     

Expression and insights on function of potassium channel TWIK-1 in mouse kidney

Renal distribution and function of TWIK-1, a member of the two-pore-domain potassium channel family, was studied in mouse kidney. TWIK-1 is expressed in apical and subapical localizations of proximal tubule and cytoplasmic sites of thin and thick ascending limbs, distal convoluted tubules and medullary collecting duct. Studies in mice lacking intact TWIK-1 (twik-1 –/–) and wild-type mice (twik-1 +/+) revealed an attenuated ability to increase renal phosphate (Pi) reabsorption and stabilize plasma Pi concentration in response to a low Pi diet in twik-1 –/– mice. Western blot analysis and immunohistochemistry for the electrogenic 3Na⁺-1HPO₄^2–-cotransporter NaPi-2a revealed a reduced reno-cortical expression in twik-1 –/– mice under these conditions. Under normal diet, twik-1 –/– mice presented lower urinary flow rates. Acute pharmacologic blockade of the vasopressin V₂-receptor revealed both an attenuated diuretic response and an attenuated internalization of aquaporin-2 in the inner medullary collecting duct in twik-1 –/– versus +/+ mice. In summary, mice deficient for TWIK-1 presented impaired regulation of (i) Pi transport in proximal tubule and (ii) water transport in medullary collecting duct. TWIK-1 may contribute to membrane trafficking/expression of transport molecules in proximal tubule and medullary collecting duct, and possibly other renal sites of expression.X. Nie and I. Arrighi have contributed equally to this work.     

Mechanism of action of eserine on DNA synthesis in the rat liver

Eserine, in a dose of 1–2 mg/kg, inhibits incorporation of radioactive precursors into rat liver DNA by 40–50%. Eserine has a direct action on DNA synthesis in the nuclei only in high concentrations (10^–5).In vitro experiments showed that DNA-synthetic activity of hepatocyte nuclei isolated from the liver of rats receiving a preliminary injection of eserine in a dose of 1 mg/kg is significantly reduced. It is concluded that the action of eserine on incorporation of radioactive precursors into rat liver DNAin vivo is localized at the level of DNA synthesis in the nuclei. A study of the action of acetylcholine on DNA-synthetic activity of isolated liver nuclei suggests that the inhibitory action of eserine on DNA synthesis is mediated through acetylcholine.Institute of Toxicology, Ministry of Health of the USSR, Leningrad. (Presented by Academician of the Academy of Medical Sciences of the USSR, S. N. Golikov.) Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 87, No. 1, pp. 24–27, January, 1979.     

Characterization of the third origin of DNA replication of the genome of insect iridescent virus type 6

The structure of the third origin of DNA replication (CIV-ori-M) of the genome (209 kbp) of Chilo iridescent virus (CIV) was determined by DNA nucleotide sequence analysis. The CIV-ori-M is located within the DNA sequences of theEcoRI CIV DNA fragment M (7 kbp; 0.310–0.345 viral map units) between the genome coordinates 0.310 (EcoRI site) and 0.317 (NcoI site). The DNA nucleotide sequence of theEcoRI/NcoI CIV DNA fragment (1601 bp) was determined for identifying the DNA sequence of the corresponding origin of DNA replication. The analysis of the DNA sequences of this region revealed the presence of a 12-mer inverted repeat at nucleotide positions 485–496 and 503–513 (485-AGATATTTGACT-496-TATGT-503-AGTCAAATATCT-513) that are able to form a hairpin-loop structure. A double-stranded DNA fragment was synthesized that corresponds to the nucleotide positions 485–513 that were cloned into the phages M13mp18 and M13mp19, and were screened for their ability to be amplified in CF-124 cell cultures infected with CIV. The successful amplification of the DNA sequence of the CIV-ori-M is strong evidence that this particular region of the CIV genome indeed serves as the origin of DNA replication.The analysis of the DNA sequence of CIV-ori-M in comparison to the DNA sequence of the two other characterized origins of DNA replication (CIV-ori-H and Y) of the CIV genome (9) was carried out, and the results are shown in Table 2. According to these data the DNA sequence homology between the DNA sequences of the CIV-ori-M and CIV-ori-Y, and between the CIV-ori-M or CIV-ori-H, was found to be 58% and 55%, respectively.     

Evidence for a gene cluster involving trichothecene-pathway biosynthetic genes in Fusarium sporotrichioides

Two overlapping cosmid clones (Cos1-1 and Cos9-1) carrying the Tox5 gene were isolated from a library of F. sporotrichioides strain NRRL 3299 genomic DNA. These cosmids were used to transform three T-2 toxin-deficient mutants that are blocked at different steps in the trichothecene pathway. Both cosmids restored T-2 toxin production to Tox3-1 ^– or Tox4-1 ^– mutants but neither restored T-2 toxin production to a Tox1–2 ^– mutant. The production of T-2 toxin by the complemented Tox3-1 ^– and Tox4-1 ^– mutants, as well as the production of diacetoxyscirpenol by the cosmid-transformed Tox1-2 ^– mutant, were 2- to 10- fold higher than in strain NRRL 3299. In addition, those transformants carrying Cos9-1 produced significantly higher levels of trichothecenes than transformants carrying Cos1-1. Two different DNA fragments (FSC13-9 and FSC14-5), representing the region of overlap between the cosmid clones, were isolated. These fragments specifically complemented either the Tox3-1 ^– mutant (FSC14-5) or the Tox4-1 ^– mutant (FSC13-9). The trichothecene-production phenotype of these transformants was similar to NRRL 3299. These results suggest that two or more genes involved in the biosynthesis of trichothecenes are closely linked to Tox5.     

Haplotypes Eco47 III-Nsp I sites frequencies on the IDUA gene in Mexican native population

Background. – The frequency of haplotypes of Nsp I–Eco47 III sites, at the IDUA (α-L iduronidase) gene, in Huichol, Tarahumara and Mestizo Mexican population is reported.Methods. – Eco47 III and Nsp I intragenic polymorphisms in IDUA gene are studied in three (Mestizo, Huichol and Tarahumara populations) Mexican groups. Data from normal Australian [Hum. Genet. 90 (1992) 327] individuals were considered for comparative analyses.Results. – The genotypes for IDUA Eco47 III and Nsp I sites in Mexicans were in agreement with Hardy–Weinberg equilibrium. Allele frequency distributions for individual sites differed (P < 0.05) except at site B₁ in the Huichol group. Haplotype Eco47 III–Nsp I frequency distributions were different in the three Mexican normal groups, and it was also observed when to compared with the normal Australians.Conclusions. – This characteristic makes the two IDUA polymorphic sites useful for identification purposes, and these polymorphisms could be included in a PCR based battery of DNA markers.     

Inhibition of thymidylate biosynthesis induces mitotic unequal sister chromatid recombination in Saccharomyces cerevisiae

Summary Inhibition of thymidylate biosynthesis has been found to induce deletion of a LEU2 insert from the ribosomal DNA gene cluster of haploid strains of Saccharomyces cerevisiae. Loss of the insert was detected phenotypically by the enhanced production of both sectored (leu⁺/leu^–) and non-sectored (leu^–) colonies. Hybridization patterns obtained by Southern blot analysis of DNA from the leu⁺ and leu^– sectors were consistent with the occurrence of unequal sister chromatid recombination. The induction of sectored colonies was prevented by the rad52-1 mutation but not by defects in RAD6. However, the formation of non-sectored leu^– colonies was induced by thymidylate depletion in both rad52-1 and rad6 strains.     

Effects of calcium channel modulators on the proliferation of mouse spleen lymphocytesin vitro

The effects of nimodipine (voltage-dependent calcium channel blocker), CGP 28392 and BAY K 8644 (novel dihydropyridine derivatives that are considered as calcium entry stimulators) on the spontaneous proliferation of mouse spleen lymphocytes were studiedin vitro. [³H]-thymidine incorporation into DNA of lymphocytes was used as an sensitive index of the cell proliferation. It has been found that nimodipine (10^–4 M–10^–6 M) significantly inhibited the [³H]-thymidine uptake in a dose dependent fashion with ED₅₀ value of 2.4×10^–5 M. Unexpectedly, CGP 28392 (10^–4 M–10^–7 M) acts as a calcium entry blocker and produces a strong inhibitory effect on lymphocyte proliferation (ED₅₀–2×10^–5 M). BAY K 8644 at a high concentration (10^–4 M) also has an inhibitory effect but at a lower concentration (10^–6 M–10^–10 M) significantly increased [³H]-thymidine uptake and abolished the inhibitory effect of nimodipine. This effect of nimodipine was also reversed by 5×10^–3 M calcium chloride. These findings indicate that calcium channel modulators can regulate the proliferation of mouse spleen lymphocytesin vitro.     

Sequence analysis of ospA genes shows homogeneity within Borrelia burgdorferi sensu stricto and Borrelia afzelii strains but reveals major subgroups within the Borrelia garinii species

The genes coding for the outer surface protein A (OspA) of 19 different Borrelia burgdorferi strains belonging to the seven OspA-serotypes 1–7, previously described [Wilske et al. (1993) J Clin Microbiol, 31: 340–350], have been investigated. B. burgdorferi sensu lato strains were chosen from various biological sources (ticks, human skin and cerebrospinal fluid) as well as different geographical origins (Germany, Slovenia, Austria, United States). The open reading frames of all ospA genes consist of 819–825 nucleotides corresponding to proteins of approximately 30 kDa. The ospA sequences obtained in this study and previous published studies were compared with the results from OspA serotyping with monoclonal antibodies. The classification into the seven OspA serotypes could be confirmed on a genetic basis (ospA genotypes 1–7) for all strains analyzed so far (n=29). In addition, one strain without OspA expression could be assigned to ospA genotype 2. Genetic stability could be proven for the ospA gene of B. burgdorferi strain PWudI after inocculation and reisolation from a gerbil. However, we found evidence for intragenic recombination by cluster analysis of ospA sequence data. Accordance of ospA genotype 1 strains with B. burgdorferi sensu stricto and ospA genotype 2 strains with B. afzelii, as well as the ospA genotype strains 3–7 with B. garinii was confirmed by pulsed-field gel electrophoresis of MluI-digested genomic DNA. B. garinii is not only more heterogenous in respect to the OspA-encoding genes, but shows moreover major subgroups formed by genotypes 4, 5 and 6 and genotypes 3 and 7, respectively. The latter group has not been described previously and is specifically recognized by an OspA-specific monoclonal antibody L32 1F7.     

Chloride channels in the luminal membrane of the rectal gland of the dogfish (Squalus acanthias)

The rectal gland of the dogfish (Squalus acanthias) secretes chloride via a chloride channel present in the apical cell membrane. Using the patch clamp technique in isolated perfused rectal gland tubules [7], two types of chloride channels are demonstrable in the apical membrane of cyclic AMP treated tubule segments. A small channel of about 11 pS and another channel of 40–50 pS are present. The small channel is described in the succeeding report. With NaCl on both sides (excised patches) the current amplitude of the larger channel is an almost linear function of the voltage (±50 mV). However, the open probability of this channel is grossly reduced at negative clamp potentials (corresponding to cell hyperpolarization). Therefore, the macroscopic Cl^– current through this channel is reduced with hyperpolarization on the cytosolic side. An analysis of time constants of this channel reveals that at depolarized voltages two open and two closed time constants of about 1 ms and of about 10 ms, respectively, are demonstrable. With hyperpolarized voltages the larger open state time constant is reduced significantly. This type of chloride channel is blocked reversibly by diphenylamine-2-carboxylate (10^–4 mol/l) and by 5-nitro-2-(3-phenylpropylamino)-benzoate (10^–5 mol/l). The channel is selective for Cl^– over Na^– and K^– as well as over Br^–. It is, however, permeable for NO ₃ ^- . Since this channel is very rare or absent in nonstimulated rectal gland tubules, it is very likely that this type of channel is responsible for hormone and cAMP dependent chloride secretion in this organ.Supported by Deutsche Forschungsgemeinschaft Gr 480 and by NSF and NIH grants to the MDIBL     

Properties of the bursting Na channel in the presence of DPI 201-106 in guinea-pig ventricular myocytes

Single Na channel currents were measured in cellattached patches of guinea-pig ventricular myocytes in the presence of the S-enantiomer of DPI 201-106. DPI changes the kinetic pattern of channel activity from short living openings at the beginning of a depolarizing pulse (voltageindependent mean open time about 0.4 ms between –60 and –20 mV), into longlasting bursts of openings. The single channel current-voltage relation can be approximated by a straight line with a single channel conductance of 15 pS, which is the same as in the absence of DPI, and a reversal potential near the estimated Na equilibrium potential (+ 74 mV). The ensemble averaged Na current shows a fast peak of inward current, which partially decays within less than 10 ms, but which shows a large component which decays very slowly with a time constant of the order of 1s (1.31±0.6 s at –30 mV, 19 measurements in 12 cell-attached patches). The slowly decaying component activates with a half-maximum potential at –55.4±2.3 mV and a slope parameter s of 4.9±1.9 mV. The half-maximum potential of the steady-state inactivation is –115.6±1.8 mV, and the slope parameter is 9.1±1.5 mV. The open time distribution can be fitted by a single exponential only at potentials negative to –40 mV. The time constant is 1.3±0.14 ms at –50 mV (7 patches). At more positive potentials a slower second component is present (14.4±7.1 ms at –30mV, 7 patches), in addition to a fast component with a time constant between 2 and 3 ms (2.2±1.9 ms at –30 mV, 7 patches). The closed time distribution contains two exponential components. The time constant of the fast component decreases from about 1.3 ms at –70 mV to 0.3 ms at –20 mV (0.3±0.17 ms at –30 mV, 10 patches). The time constant of the second component decreases from about 4.0 ms at –70 mV to 1.5 ms at –20 mV (2.2±1.8 ms at –30 mV, 10 patches). At potentials positive to –50 mV also a small very slow component is present (8.8±5.6 ms at –30 mV). The contribution of the slow component to the closed time distribution increases for stronger depolarizations from 2.2±2.1% at –40 mV to 64±22% at –20 mV. The contribution of short closings to the total number of closings is increased from 51±4% at –70 mV to 83±12% at –20 mV. Openings occur clearly in bursts and the burst duration shows a very good correlation with the time constant of the decay of the ensemble average current in a large number of different experimental conditions. The channels are blocked by application of TTX. With 10 and 30 M TTX in the patch pipette (1 s pacing interval) the probability of the channel being open is decreased by about 90%. The long mean open time is significantly reduced when TTX is present in the patch pipette (5.7±2.6 ms at –30 mV with 5–30 M TTX, 4 patches,n=11). No significant differences are obtained for both closed times (_c1 = 0.26±0.07 ms, _c2 = 1.9±0.7 ms, 4 patches,n=9). The steady-state inactivation is not significantly changed in the presence of TTX (V_H=–112±2.9 mV, s=9.0±2.8 mV). The open state probability of the bursting Na channel is also reduced by more than 80% when 200 M Cd is present in the pipette solution (all mean ±SD).     

Nucleotide sequence relationships of double-stranded RNAs in flax rust,Melampsora lini

Flax rust, Melampsora lini strain SP6, contains 11 double-stranded (ds) RNA molecules with a total length of about 25 kbp. The dsRNAs are inherited in three genetic units: the L unit comprising a single 5.2 kbp dsRNA and contained within a 40-nm virus-like particle, and the A and B units each consisting of five dsRNAs (A1–A5, and B1–B5) ranging in size from 1.2 to 2.7 kbp. This paper reports the isolation of a cDNA library representing 10 of the 11 dsRNAs. By nucleic-acid hybridization techniques it has been shown that all ten sequences are unique showing no detectable cross-hybridization with any other dsRNA present in the rust. A near fulllength sequence of 1932 bp of the B3 dsRNA is reported and contains several open reading frames, the largest of which comprises most of the molecule.     

Properties and regulation of chloride channels in cystic fibrosis and normal airway cells

The present study examines the properties of Cl^–channels in cultured respiratory cells of cystic fibrosis (CF) patients and normal (N) individuals. In excised membrane patches the conductances for CF and N Cl^– channels were larger at positive as compared to negative clamp voltages (V _c): 74±2.6 (V _c > 0) and 47±2.0 pS (V _c < 0) for CF (n= 57) and 69±3.6 (V _c > 0) and 45±2.3 pS (V _c < 0) for N (n=35). The open probability (P _o) of the channel increased markedly with depolarization. Both the voltage dependence of the conductance and of P _o contribute to the outward rectification of the channel. The time histogram analysis reveals two open and two closed time constants. The selectivity of the channel was Cl^–=Br^– =I^– > NO ₃ ^– gluconate. The channel was inhibited reversibly by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) at 10^–7 mol/l to 10^–5 mol/l. While Cl^– channels were present in cell attached patches of N cells, they were absent in those of CF cells. The mean conductance for cell attached (N) Cl^– channels was 76±3.2 pS for positive clamp voltages (V _c) and 46±3.9 pS for negative V _c (n=8). When the membrane patches were excised from CF cells Cl^– currents appeared spontaneously (n=19). The immediate appearance (within 1 s) of Cl^– channels after excision was observed at positive (n=6) as well as at negative clamp voltage (n=13). Excision activation of CF Cl^– channels was observed at low (< 10^–9 mol/l) or high (10^–3 mol/l) calcium activities on the cytosolic side of the excised patch. Variation of the Ca⁺ activity (< 10^–9–10^–3 mol/l) or pH (6.5–8.5) on the cytosolic side exerted no effects on these Cl^– channels. These results suggest that Cl^– channels are present in the apical membrane of CF and N respiratory cells but they seem to be inhibited in intact CF cells. Excision of the patch and hence removal of the cytosolic inhibitor leads to an activation of Cl^– channels. The Cl^– channels in excised patches of N and CF cells have identical properties.