Use of competitive DNA hybridization to identify differences in the genomes of bacteria

Although recent technological advances in DNA sequencing and computational biology now allow scientists to compare entire microbial genomes, comparisons of closely related bacterial species and individual isolates by whole-genome sequencing approaches remains prohibitively expensive for most laboratories. Here we report the development and testing of a biochemical approach for targeted sequencing of only those chromosomal regions that differ between two DNA preparations. The method, designated GFE (genome fragment enrichment) uses competitive solution hybridization and positive selection to obtain genomic DNA fragments that are present in one pool of fragments but not another. Repeated comparisons of the genomes of Enterococcus faecalis and E. faecium led to the identification of 225 putative genome-specific DNA fragments. Species and strain variations within these fragments were confirmed by both experimental and bioinformatic analyses. The E. faecalis genome-specific sequences identified included both a preponderance of those predicted to encode surface-exposed proteins, as well as several previously described unique marker regions embedded within highly conserved rrn operons. The GFE strategy we describe efficiently identified genomic differences between two enterococcal genomes, and will be widely applicable for studying genetic variation among closely related bacterial species.     

Role of vinculin in regulating focal adhesion turnover

Although vinculin (-/-) mouse embryo fibroblasts assemble focal adhesions (FAs), they spread more slowly, less extensively, and close a wound more rapidly than vinculin (+/+) cells. To investigate the structure and dynamics of FAs in these cells, we used real-time interference reflection microscopy (IRM) thus avoiding the need to express exogenous GFP-tagged FA proteins which may be misregulated. This showed that the FAs were smaller, less abundant and turned over more rapidly in vinculin null compared to wild-type cells. Expression of vinculin rescued the spreading defect and resulted in larger and more stable FAs. Phosphatidylinositol 4,5-bisphosphate (PIP2) is thought to play a role in vinculin activation by relieving an intramolecular association between the vinculin head (Vh) and tail (Vt) that masks the ligand binding sites in Vh and Vt. To investigate the role of the vinculin/PIP2 interaction in FA dynamics, we used a vinculin mutant lacking the C-terminal arm (residues 1053-1066) and referred to as the deltaC mutation. This mutation reduced PIP2 binding to a Vt deltaC polypeptide by >90% compared to wild type without affecting binding to Vh or F-actin. Interestingly, cells expressing the vinculin deltaC mutant assembled remarkably stable FAs. The results suggest that vinculin inhibits cell migration by stabilising FAs, and that binding of inositol phospholipids to Vt plays an important role in FA turnover.     

Passive immunization of lactating mice with stanniocalcin-1 antiserum reduces mammary gland development, milk fat content, and postnatal pup growth

During pregnancy and lactation in rodents, stanniocalcin-1 (STC-1) production by the ovaries is upregulated markedly and released into the circulation. The mammary glands are one target of this systemically delivered hormone. The purpose of this study was to lower serum levels of STC-1 in lactating mice through passive immunization so as to monitor the effects on mammary gland function and postnatal pup growth. Passive immunization significantly reduced circulating hormone levels, and pup growth was significantly compromised (30%), even though control and experimental litters had ingested equal amounts of milk. When mammary glands were analyzed, the alveolar area was significantly reduced in antibody-treated mothers. An analysis of milk composition revealed no changes in lactose, protein, or electrolyte levels but an approximately 40% reduction in triglyceride levels. The latter was due to a significant reduction in mammary gland lipoprotein lipase activity and led to a significant buildup of triglycerides in the serum. Body fat content was also significantly reduced in pups from antibody-treated mothers, whereas pup fecal fat content was increased. In mothers, passive immunization also caused significant behavioral effects, in particular, increased locomotor and hindleg rearing activities. Collectively, the results suggest that systemically derived STC-1 has important effects on mammary gland development and the transfer of serum-based triglycerides into milk. Locomotor effects suggest that STC-1 also has a role in maternal behavior.     

A reassessment of copper(II) binding in the full-length prion protein

It has been shown previously that the unfolded N-terminal domain of the prion protein can bind up to six Cu2+ ions in vitro. This domain contains four tandem repeats of the octapeptide sequence PHGGGWGQ, which, alongside the two histidine residues at positions 96 and 111, contribute to its Cu2+ binding properties. At the maximum metal-ion occupancy each Cu2+ is co-ordinated by a single imidazole and deprotonated backbone amide groups. However two recent studies of peptides representing the octapeptide repeat region of the protein have shown, that at low Cu2+ availability, an alternative mode of co-ordination occurs where the metal ion is bound by multiple histidine imidazole groups. Both modes of binding are readily populated at pH 7.4, while mild acidification to pH 5.5 selects in favour of the low occupancy, multiple imidazole binding mode. We have used NMR to resolve how Cu2+ binds to the full-length prion protein under mildly acidic conditions where multiple histidine co-ordination is dominant. We show that at pH 5.5 the protein binds two Cu2+ ions, and that all six histidine residues of the unfolded N-terminal domain and the N-terminal amine act as ligands. These two sites are of sufficient affinity to be maintained in the presence of millimolar concentrations of competing exogenous histidine. A previously unknown interaction between the N-terminal domain and a site on the C-terminal domain becomes apparent when the protein is loaded with Cu2+. Furthermore, the data reveal that sub-stoichiometric quantities of Cu2+ will cause self-association of the prion protein in vitro, suggesting that Cu2+ may play a role in controlling oligomerization in vivo.     

Novel role for the C terminus of Saccharomyces cerevisiae Rev1 in mediating protein-protein interactions

The Saccharomyces cerevisiae REV3/7-encoded polymerase zeta and Rev1 are central to the replicative bypass of DNA lesions, a process called translesion synthesis (TLS). While yeast polymerase zeta extends from distorted DNA structures, Rev1 predominantly incorporates C residues from across a template G and a variety of DNA lesions. Intriguingly, Rev1 catalytic activity does not appear to be required for TLS. Instead, yeast Rev1 is thought to participate in TLS by facilitating protein-protein interactions via an N-terminal BRCT motif. In addition, higher eukaryotic homologs of Rev1 possess a C terminus that interacts with other TLS polymerases. Due to a lack of sequence similarity, the yeast Rev1 C-terminal region, located after the polymerase domain, had initially been thought not to play a role in TLS. Here, we report that elevated levels of the yeast Rev1 C terminus confer a strong dominant-negative effect on viability and induced mutagenesis after DNA damage, highlighting the crucial role that the C terminus plays in DNA damage tolerance. We show that this phenotype requires REV7 and, using immunoprecipitations from crude extracts, demonstrate that, in addition to the polymerase-associated domain, the extreme Rev1 C terminus and the BRCT region of Rev1 mediate interactions with Rev7.     

Modelling environmental variation in Young's modulus for Pinus radiata and implications for determination of critical buckling height

• Background and Aims Although density-specific stiffness,E/, (where E is Young's modulus and is wood density) is oftenassumed constant by the elastic similarity model, and in determinationof critical buckling height (H_crit), few studies have testedthis assumption within species. Here this assumption is testedfor Pinus radiata growing across an environmental gradient,and theory is combined with data to develop a model of Young'smodulus. • Methods Analyses use an extensive series of environmentalplots covering the range of climatic and edaphic conditionsover which P. radiata is grown in New Zealand. Reduced majoraxis regression was used to determine scaling exponents betweenlog–log plots of H_crit vs. groundline diameter (D), andE/ vs. D. Path analysis was used to identify significant directand indirect (through stem slenderness) edaphic and climaticinfluences on E. • Key Results Density-specific stiffness exhibited 3-foldvariation. As E/ scaled positively with D, the exponent of 0·95between H_crit and D exceeded the assumed value of 0·67under constant E/. The final path analysis model included meanair temperature in early autumn (T_aut) and slenderness as significant(P < 0·05) positive direct influences on E. Tree leafarea index and T_aut were indirectly associated with E throughtheir significant (P < 0·05) positive direct relationshipwith stem slenderness. Young's modulus was most sensitive toT_aut, followed by stem slenderness then leaf area index, andthe final model explained 76 % of the variance in E. • Conclusions The findings suggest that within speciesE/ variation may influence H_crit and the scaling exponent betweenD and H_crit so important in assumptions regarding allometricrelationships. The model presented may provide a useful meansof determining variation in E, E/ and H_crit across environmentalgradients.     

Identification of new differentiation inducing factors from Dictyostelium discoideum

Developing Dictyostelium discoideum amoebae form a stalked fruiting body in which individual cells differentiate into either stalk cells or spores. The major known inducer of stalk cell differentiation is the chlorinated polyketide DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one); however a mutant blocked in the terminal step of DIF-1 biosynthesis still produces one of the prestalk cell subtypes – the pstA cells – as well as some mature stalk cells. We therefore searched for additional stalk cell-inducing factors in the medium supporting development of this mutant. These factors were purified by solvent extraction and HPLC and identified by mass spectroscopy and NMR. The mutant lacked detectable DIF-2 and DIF-3 (the pentanone and deschloro homologues of DIF-1) but four major stalk cell-inducing activities were detected, of which three were identified. Two compounds were predicted intermediates in DIF-1 biosynthesis: the desmethyl, and desmethyl-monochloro analogues of DIF-1 (dM-DIF-1 and Cl-THPH, respectively), supporting the previously proposed pathway of DIF-1 biosynthesis. The third compound was a novel factor and was identified as 4-methyl-5-pentylbenzene-1,3-diol (MPBD) with the structure confirmed by chemical synthesis. To investigate the potential roles of these compounds as signal molecules, their effects on morphological stalk and spore differentiation were examined in cell culture. All three induced morphological stalk cell differentiation. We found that synthetic MPBD also stimulated spore cell differentiation. Now that these factors are known to be produced and released during development, their biological roles can be pursued further.     

An unusual glucoside from Cleistanthus gracilis

Extraction of roots and stems of Cleistanthus gracilis furnished common triterpenes, plant sterols and the unusual glucoside (+) gracicleistanthoside, the glucoside of 2-beta-hydroxy-8-azabicyclo-(5,2,0)-4beta,9beta-epoxynona-5,7-diene.     

Normative nerve conductions in the tail of rhesus macaques (Macaca mulatta)

BACKGROUND: The aim of this study was to test the feasibility of using the tail of Macaca mulatta for neurophysiological testing of the peripheral nervous system. METHODS: Motor and sensory nerve conduction studies (NCS) of the tail were obtained by surface stimulation and recording. The technique utilized was novel. Unlike other NCS obtained from other peripheral nerves, this technique did not require any special neurophysiological expertise. RESULTS: The latency of the motor and sensory response was 2.5 +/- 0.71 and 1.1 +/- 0.27 ms respectively. The amplitude of the motor and sensory response was 8.1 +/- 5.1 mV and 14.6 +/- 9.4 microV respectively. Similar to human beings, there was a statistically significant relationship between age and motor amplitude, motor latency and sensory latency. CONCLUSIONS: Based on our results, a relatively simple, reproducible neurophysiological monitoring technique of the peripheral nervous system is possible.