NMR investigations of duplex stability of phosphorothioate and phosphorodithioate DNA analogues modified in both strands.

Duplex formation from the self-complementary 12mer d(CGCGAATTCGCG) (Dickerson dodecamer) in which all phosphodiester linkages were replaced by phosphorothioate or phosphorodithioate linkages was studied using variable-temperature 1H and 31P NMR spectroscopy. Melting temperatures of the dodecamer, measured spectrophotometrically, showed significant decrease upon sulfur substitution (Tm 49 degrees C for the phosphorothioate and 21 degrees C for the phosphorodithioate, compared with 68 degrees C for the unmodified oligomer, in 1 M salt). Hyperchromicity observed upon melting of the dithioate was surprisingly low. NOESY spectra of the monothioate showed a cross-peak pattern characteristic for a right-handed duplex. Imino proton resonances of the duplex, shown by the mono- and the dithioate, were similar to those of the parent compound. In spite of monophasic melting curves, temperature dependence of the imino proton resonances and phosphorus resonances of the phosphorodithioate indicated heterogeneity with respect to base-pairing, compatible with the presence of a hairpin loop. Relaxation times (T1) of the imino protons in the phosphorothioate, determined by the saturation recovery method, were considerably shorter than in the unmodified oligomer. Base-pair lifetimes in the unmodified Dickerson dodecamer, determined by catalyst-dependent changes in relaxation rates of imino protons, were in the range of 2-30 ms at 20 degrees C. Strongly reduced base-pair lifetimes were found in the phosphorothioate analogue.     

A mutant cysteinyl-tRNA synthetase affecting timing of chromosomal replication initiation in B. subtilis and conferring resistance to a protein kinase C inhibitor.

A Bacillus subtilis mutant spnA95 was isolated as resistant at 30 degrees C to the protein kinase C (PKC) inhibitor, sphinganine, and temperature sensitive for growth. As deduced by flow cytometry measurements, the mutant has a 35% reduced initiation mass at permissive temperature, resulting in initiation of DNA replication much earlier in the cell cycle than in the wild type. This modification is accompanied by a change in cell size, as determined by phase-contrast microscopy and flow cytometry. Therefore, this strain displays the characteristics of a novel cell clock mutant. spnA is a newly identified gene in B.subtilis and was shown to encode a cysteinyl-tRNA synthetase. At non-permissive temperature, the mutant was defective in the synthesis of P70, a protein with several characteristics of PKC (a cysteine-rich protein). As one possibility, we propose that the altered timing of replication may be due to the reduced synthesis of specific cysteine-rich proteins normally involved in controlling chromosomal replication initiation in B. subtilis.     

Attempts to define functional domains of gap junction proteins with synthetic peptides.

To map the binding sites involved in channel formation, synthetic peptides representing sequences of connexin 32 were tested for their ability to inhibit cell-cell channel formation. Both large peptides representing most of the two presumed extracellular loops of connexin32 and shorter peptides representing subsets of these larger peptides were found to inhibit cell-cell channel formation. The properties of the peptide inhibition suggested that the binding site is complex, involving several segments of both extracellular loops. One of the peptides (a 12-mer) did not inhibit but instead was found to form channels in membranes. Both in oocyte membranes and in bilayers, the channels formed by the peptide were asymmetrically voltage dependent. Their unit conductances ranged from 20 to 160 pS. These data are discussed in the form of a model in which the connexin sequence represented by the peptide is part of a beta structure providing the lining of the channel pore.     

Purification and characterization of the phospho-alpha(1,1)glucosidase (TreA) of Bacillus subtilis 168.

The intracellular phospho-alpha(1,1)glucosidase TreA from Bacillus subtilis has been overproduced in Escherichia coli and purified by ion-exchange chromatography and gel filtration. The molecular mass, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 64 kDa. Isoelectric focusing indicated homogeneity of the protein, and its pI was determined to be 4.3. Characterization of the enzyme showed a protein which is stable up to 44 degrees C after temperature treatment for 15 min. The temperature optimum was found to be 37 degrees C, and the pH optimum was 4.5. TreA activity is stimulated by high salt concentrations with different efficiencies depending on the kind of salt. When increasing amounts of ammonium sulfate are used, the increase of TreA activity is correlated with a conformational change of the protein or dimerization. The substrate specificity of the purified enzyme was characterized, showing additionally that trehalose is also hydrolyzed, but to a much smaller extent than trehalose-6-phosphate. In vitro, the presence of glucose reduces TreA activity, indicating product inhibition of the enzyme.     

Effects of fish predation and habitat type on stream benthic communities

The influence of habitat on interactions between a fish predator (brown trout Salmo trutta) and a benthic invertebrate community was studied in nine field enclosures (8 ×3 m) in a creek in southern Sweden. Three habitat treatments were tested, a shallow sandy habitat, a deep habitat containing a mixture of large and small cobbles and a moderately deep habitat with large cobbles. The one month-long experiment showed that there were no major differences in the abundance and biomass of the benthic macroinvertebrate fauna among these habitats as no functional groups of invertebrates and only a few taxa differed between treatments. Invertebrate drift rates decreased over time, which was probably related to seasonal changes in invertebrate life cycles or to effects of predation independent of habitat type, as there was no difference between treatments.     

X-linked progressive mixed deafness: a new microdeletion that involves a more proximal region in Xq21.

We report a large two-generation pedigree with seven affected males segregating for an X-linked mixed conductive sensorineural deafness. The patients present with atypical Mondini-like dysplasia, dilated petrous facial canal, dilatation of the internal auditory meatus fully connected with enlarged cochlear canals, and, in one patient, a wide bulbous posterior labyrinth. Obligatory carrier females are mildly affected. Molecular characterization of this family revealed a deletion of locus DXS169, in Xq21.1. Loci DXS72 and DXS26, which, respectively, flank DXS169 proximally and distally, were intact. Since a gene responsible for X-linked progressive mixed deafness with perilymphatic gusher (DFN3) has previously been assigned by deletion mapping to a slightly more distal interval between DXS26 and DXS121, this study indicates either two different deafness genes or the involvement of a very large region in Xq21.     

A topoisomerase II cleavage site is associated with a novel mitochondrial DNA deletion

Mitochondrial myopathies and encephalopathies can be caused by nucleotide substitutions, deletions or duplications of the mitochondrial DNA (mtDNA). In one such disorder, Kearns-Sayre Syndrome (KSS), large-scale hetero-plasmic mtDNA deletions are often found. We describe a 14-year-old boy with clinical features of KSS, plus some additional features. Analysis of the entire mitochondrial genome by the polymerase chain reaction and Southern blotting revealed a 7864-bp mtDNA deletion, heteroplasmic in its tissue distribution. DNA sequencing established that the deletion was between nucleotides 6238 and 14103, and flanked by a 4-bp (TCCT) direct repeat sequence. Deletions between direct repeats have been hypothesised to occur by a slipped-mismatching or illegitimate recombination event, or following the DNA cleavage action of topoisomerase II. Analysis of the gene sequence in the region surrounding the mtDNA deletion breakpoint in this patient revealed the presence of putative vertebrate topoisomerase II sites. We suggest that direct repeat sequences, together with putative topoisomerase II sites, may predispose certain regions of the mitochondrial genome to deletions.