Short-Term,Intermittent Fasting Induces Long-Lasting Gut Health and TOR-Independent Lifespan Extension

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Novel post-replicative DNA modification in Streptomyces: analysis of the preferred modification site of plasmid pIJ101.

Both Streptomyces lividans and Streptomyces avermitilis have the ability to site specifically modify their DNA, rendering it susceptible to in vitro Tris-dependent double-strand cleavage. We have cloned a 160 bp fragment containing the preferred modification site of plasmid pIJ101 and, employing an in vitro primer extension assay, determined that the modifications occur at guanine residues on either strand separated by 3 bp. These guanines are located within a 6 bp palindromic 'core' sequence. A cloned copy of a 35 bp region of the plasmid containing this core sequence was not recognized by the modifying activity in vivo. To further investigate the nature of the site specificity a set of deletion mutants of the 160 bp sequence were analysed. This revealed that a substantial portion of this sequence is essential for authentic modification. The essential region contains three 13 bp direct repeats, the central one containing the core sequence, while the left-hand and right-hand copies overlap two potential stem-loop structures. Deletion of either left- or right-hand repeat structures abolishes modification within the core sequence, although the left-hand deletion resulted in modification at a secondary site within the right-hand direct repeat. These data support a post-replicative mechanism of modification, underlined by the observation that the modifications are not detected in single-stranded plasmid replication intermediates.     

Novel site-specific DNA modification in Streptomyces: analysis of preferred intragenic modification sites present in a 5.7 kb amplified DNA sequence.

Both Streptomyces lividans and Streptomyces avermitilis encode similar systems of post-replicative DNA modification which act site-specifically on closely opposed guanines on either strand. The modifications can be detected since they react in vitro with an oxidative derivative of Tris, resulting in strand cleavage. Previous analysis of the preferred modification site of plasmid pIJ101 indicated that extensive amounts of flanking sequence, including direct and inverted repeat structures, are required to direct modification in vivo within a central 6 bp palindrome. We have now examined the preferred modification sites of a chromosomal element, the 5.7 kb amplified DNA sequence (ADS5.7) found in certain S. lividans mutants. In contrast to the pIJ101 site, each of the ADS5. 7sites is intragenic and modified with a 10-fold reduced frequency. However, similar extents of flanking sequence are required for authentic double-strand modification; deletion mutants exhibited different modification profiles, including displaced double-stranded or single-stranded modi-fication. Comparison of different modification sites reveals conservation of the central core sequence, but no significant similarities between flanking sequences. Enhanced modification was detected in a cloned region of the ADS5.7, suggesting that local DNA topology, probably influenced by both DNA supercoiling and the nature of flanking sequences, can influence the modifying activity.     

Absolute hearing thresholds and critical masking ratios in the European barn owl: a comparison with other owls

Absolute thresholds and critical masking ratios were determined behaviorally for the European barn owl (Tyto alba guttata). It shows an excellent sensitivity throughout its hearing range with a minimum threshold of −14.2 dB sound pressure level at 6.3 kHz, which is similar to the sensitivity found in the American barn owl (Tyto alba pratincola) and some other owls. Both the European and the American barn owl have a high upper-frequency limit of hearing exceeding that in other bird species. Critical masking ratios, that can provide an estimate for the frequency selectivity in the barn owl's hearing system, were determined with a noise of about 0 dB spectrum level. They increased from 19.1 dB at 2 kHz to 29.2 dB at 8 kHz at a rate of 5.1 dB per octave. The corresponding critical ratio bandwidths were 81, 218, 562 and 831 Hz for test-tone frequencies of 2, 4, 6.3 and 8 kHz, respectively. These values indicate, contrary to expectations based on the spatial representation of frequencies on the basilar papilla, increasing bandwidths of auditory filters in the region of the barn owl's auditory fovea. This increase, however, correlates with the increase in the bandwidths of tuning curves in the barn owl's auditory fovea. Accepted: 27 November 1997     

NMR characterization of the metallo-beta-lactamase from Bacteroides fragilis and its interaction with a tight-binding inhibitor: role of an active-site loop.

Understanding the structure and dynamics of the enzymes that mediate antibiotic resistance of pathogenic bacteria will allow us to take steps to combat this increasingly serious public health hazard. Complete backbone NMR resonance assignments have been made for the broad-specificity metallo-beta-lactamase CcrA from Bacteroides fragilis in the presence and absence of a tight-binding inhibitor. Chemical shift indices show that the secondary structure of the CcrA molecule in solution is very similar to that in published crystal structures. A loop adjacent to the two-zinc catalytic site exhibits significant structural variation in the published structures, but appears from the NMR experiments to be a regular beta-hairpin. Backbone heteronuclear NOE measurements indicate that this region has slightly greater flexibility on a picosecond to nanosecond time scale than the molecule as a whole. The loop appears to have an important role in the binding of substrates and inhibitors. Binding of the inhibitor 3-[2'-(S)-benzyl-3'-mercaptopropanoyl]-4-(S)-carboxy-5, 5-dimethylthiazolidine causes a marked increase in the stability of the protein toward unfolding and aggregation, and causes changes in the NMR resonance frequencies of residues close to the active (zinc-binding) site, including the beta-hairpin loop. There is a small but significant increase in the heteronuclear NOE for this loop upon inhibitor binding, indicative of a decrease in flexibility. In particular, the NOE of the indole ring of tryptophan 49, at the tip of the beta-hairpin loop, changes from a low value characteristic of a random coil chain to a significantly higher value, close to that observed for the backbone amides in this region of the protein. These results strongly suggest that the hairpin loop participates in the binding of substrate and in the shielding of the zinc sites from solvent. The broad specificity of the CcrA metallo-beta-lactamase may in fact reside in the plasticity of this part of the protein, which allows it to accommodate and bind tightly to substrates of a variety of shapes and sizes.     

Single cells can sense their position in a morphogen gradient.

Xenopus blastula cells show a morphogen-like response to activin by expressing different genes according to the concentration of activin to which they are exposed. To understand how cells recognize their position in a concentration gradient, it is essential to know whether each cell responds individually to activin concentration. An alternative idea, proposed by previous work, is that cells need to interact with their neighbours to generate a concentration-related response. To distinguish between these ideas, we have cultured blastula cells under conditions which provide different degrees of contact with other cells, allowing nil to maximum communication with their neighbours. The cultures include cells attached to fibronectin and cells resting unattached on an agarose base. The cultures also include cells that have no contact with any cell except their clonal progeny, cells that have lateral contact to neighbouring cells, and cells that are completely enveloped by other cells in a reaggregate. We have used RNase protection and in situ hybridization to assay the expression of the activin-responsive Xenopus genes Xbra, Xgsc, Xeomes, Xapod, Xchordin, Mix1, Xlim1 and Cerberus. We find no difference in gene expression between cells attached to fibronectin and those unattached on agarose. Most importantly, we find that cells respond to activin in a concentration-related way irrespective of their degree of contact with other cells. Therefore interaction among cells is not required for the interpretation of morphogen concentration, at least in the case of the early genes studied here. We conclude that isolated blastula cells can sense and respond individually to activin by expressing genes in a concentration-dependent way.     

Characterization of monomeric and dimeric B domain of Staphylococcal protein A.

Both monomeric and dimeric constructs of the B domain of protein A from Staphylococcus aureus have been characterized by NMR, CD and fluorescence spectroscopy. The monomeric form of the protein was synthesized using a novel method incorporating the use of a recombinant, folded, chimeric protein. A comparison of the recombinant monomeric form with the commercially available dimeric form indicates that, although the dimer retains the integrity of the three-helix bundle structure present in the monomer, there are interdomain contacts in the dimeric form. A single long-lived water molecule in the hydrophobic core of the three-helix bundle of monomeric protein A may represent an important stabilizing factor for the three-helix bundle topology.