The Orf18 gene product from conjugative transposon Tn916 is an ArdA antirestriction protein that inhibits type I DNA restriction-modification systems

Gene orf18, which is situated within the intercellular transposition region of the conjugative transposon Tn916 from the bacterial pathogen Enterococcus faecalis, encodes a putative ArdA (alleviation of restriction of DNA A) protein. Conjugative transposons are generally resistant to DNA restriction upon transfer to a new host. ArdA from Tn916 may be responsible for the apparent immunity of the transposon to DNA restriction and modification (R/M) systems and for ensuring that the transposon has a broad host range. The orf18 gene was engineered for overexpression in Escherichia coli, and the recombinant ArdA protein was purified to homogeneity. The protein appears to exist as a dimer at nanomolar concentrations but can form larger assemblies at micromolar concentrations. R/M assays revealed that ArdA can efficiently inhibit R/M by all four major classes of Type I R/M enzymes both in vivo and in vitro. These R/M systems are present in over 50% of sequenced prokaryotic genomes. Our results suggest that ArdA can overcome the restriction barrier following conjugation and so helps increase the spread of antibiotic resistance genes by horizontal gene transfer.     

Experimental and computational analyses of the energetic basis for dual recognition of immunity proteins by colicin endonucleases

Colicin endonucleases (DNases) are bound and inactivated by immunity (Im) proteins. Im proteins are broadly cross-reactive yet specific inhibitors binding cognate and non-cognate DNases with K_d values that vary between 10^− 4 and 10^− 14 M, characteristics that are explained by a ‘dual-recognition’ mechanism. In this work, we addressed for the first time the energetics of Im protein recognition by colicin DNases through a combination of E9 DNase alanine scanning and double-mutant cycles (DMCs) coupled with kinetic and calorimetric analyses of cognate Im9 and non-cognate Im2 binding, as well as computational analysis of alanine scanning and DMC data. We show that differential ΔΔGs observed for four E9 DNase residues cumulatively distinguish cognate Im9 association from non-cognate Im2 association. E9 DNase Phe86 is the primary specificity hotspot residue in the centre of the interface, which is coordinated by conserved and variable hotspot residues of the cognate Im protein. Experimental DMC analysis reveals that only modest coupling energies to Im9 residues are observed, in agreement with calculated DMCs using the program ROSETTA and consistent with the largely hydrophobic nature of E9 DNase-Im9 specificity contacts. Computed values for the 12 E9 DNase alanine mutants showed reasonable agreement with experimental ΔΔG data, particularly for interactions not mediated by interfacial water molecules. ΔΔG predictions for residues that contact buried water molecules calculated using solvated rotamer models met with mixed success; however, we were able to predict with a high degree of accuracy the location and energetic contribution of one such contact. Our study highlights how colicin DNases are able to utilise both conserved and variable amino acids to distinguish cognate from non-cognate Im proteins, with the energetic contributions of the conserved residues modulated by neighbouring specificity sites.     

Rapid learning of shelter position in an intertidal fish, the shanny Lipophrys pholis L.

The homing ability of an intertidal fish, the shanny Lipophrys pholis , was investigated using two experiments that were based on the shanny's natural propensity to home to a refuge. A displacement experiment demonstrated that the fish were able to accurately locate the previous position of a refuge once the shelter itself had been removed so that it could not be used as a cue to directly signal the goal location. This shows that the shanny can encode information about its familiar surroundings into a spatial map and use this information to home. A second experiment in which the cues internal and external to the experimental tank were put in conflict with one another suggested that the shanny can encode cues that are both intra- and external-tank cues in its representation of space, but that there is individual variation in the type of cues that are used, or memorized.     

Comparison of three DNA extraction methods for Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium subsp. avium

Aims:  To compare three methods for DNA extraction from Mycobacterium bovis , Mycobacterium tuberculosis and Mycobacterium avium subsp. avium .
Methods and Results:  The DNA was extracted from mycobacterial cultures using enzymatic extraction, combined bead beating and enzymatic extraction and cetyltrimethylammonium bromide (CTAB) extraction. The yield and quality of DNA were compared by spectrophotometry, agarose gel electrophoresis, restriction endonuclease analysis and PCR. The combined bead beating and enzymatic extraction method yielded more DNA. However, that method produced some sheared DNA, visible either by agarose gel electrophoresis or by restriction endonuclease analysis. All methods were appropriate for PCR amplification of a 123 bp fragment of IS 6110 in M. bovis and M. tuberculosis , and of a 1700 bp fragment of FR300 region in M. avium avium .
Conclusions:  Combined bead beating and enzymatic extraction method was the most efficient and easy method for extracting DNA from bacteria of the M. tuberculosis complex.
Significance and Impact of the Study:  The results reveal important differences among the DNA extraction methods for mycobacteria, which are relevant for the success of further downstream molecular analysis.     

斑马鱼二价体制备与多重带显带的方法学探讨

采用碱性低渗结合高氯仿一步显带技术获得了斑马鱼二价体高分辨多重G带,所出现的带纹丰富,反差明显。采用地高辛为标记的以限制性内切酶AfuⅠ介导的原位切口平移技术使斑马鱼二价体上呈现出类G带的多重带,获得了明暗反差强烈的带纹结果。通过比较多个不同分裂相的多条染色体,发现带纹的分布具明显特征性．并且特征一致,带纹数目基本吻合。首次从方法学上对斑马鱼二价体的制备过程及多重带显带技术进行了分析、探讨和总结,力求将该技术程序化、系统化,使其具有可重复性和可操作性,并对显带的可能机理进行了讨论。     

人工雌核发育草鱼近交F1代线粒体DNA限制性内切酶分析

结合差速离心法、饱和酚／氯仿／异戊醇抽提等技术．提取并纯化了雌核发育草鱼近交F．代的线粒体DNA(mtDNA)．用8种限制性内切酶对mtDNA酶切分析．结果表明：雌核发育草鱼近交Fl代mtDNA分子大小为16、77kb,除无sail酶切位点外,其余7种酶EcoRI、BalI、。YbaI、BarnHI、PstI、XhoI各产生4、4、3、3、3、2、2个切点。与已报道的普通草鱼一致．雌核发育草鱼近交F1代与普通草鱼间未检测出限制性片断长度多态性差异、这表明,雌核发育草鱼F。代与普通草鱼mtDNA遗传结构具有同一性、     

Genetic variation in two populations of the rough‐skinned newt (Taricha granulosa) assessed using novel tetranucleotide microsatellite loci

Seven novel tetranucleotide microsatellite loci were identified from a partial genomic DNA library, enriched for GATA‐motif microsatellites, from the rough‐skinned newt (Taricha granulosa). All loci were polymorphic, and one displayed a high frequency null allele. A related species, T. rivularis, displays strong site fidelity and detectable population structure over small spatial scales, so we assessed genetic variation in two samples of T. granulosa separated by 16 km. Distributions of allele frequencies differ significantly between our two sites, but small values of F_ST and Rho_ST suggest that the populations are linked by a large amount of gene flow.     

Migratory behaviour of adult pikeperch (Stizostedion lucioperca) in a lowland river

The behaviour of radio-tagged adult pikeperch (Stizostedion lucioperca (L.)) from two areas in the Danish River Gudenaa were recorded prior to, and during the spawning period. Eight of 13 tagged fish in the lower reaches of the river were located throughout the whole study. Five of these fish moved upstream to various sites in the river prior to spawning, which occurred from late April to June. The three remaining fish moved to the fjord. These movements were interpreted as a spawning migration, and it is suggested that the lower reaches of the River Gudenaa constitute an over-wintering area for pikeperch, which use different spawning areas. Ten pikeperch caught just downstream of an impassable hydropower plant were radio-tagged and translocated upstream of the dam to a reservoir. Within a week, half of the fish moved to a lake situated more than 30 km upstream the reservoir. This behaviour is hypothesised to be a homing response. The study reveals that the pikeperch is a highly mobile species with a complicated migration pattern, even in relatively small river systems.     

Atlantic salmon straying from the River Imsa

Mean estimated straying rate for Atlantic salmon Salmo salar L. leaving the River Imsa as smolts during 1976–1999 was 15% for hatchery fish and 6% for wild conspecifics. Hatchery Atlantic salmon selected for production traits during four or more generations strayed >50%. The straying rate was higher for Atlantic salmon staying 2 rather than 1 year at sea before attaining maturity. For spawning, 96% of the strays entered streams within 420 km from the River Imsa, and c . 80% entered streams within 60 km of the mouth of the River Imsa, whether the fish were wild or hatchery released. Within the 60 km zone, the number of strays caught in a river increased with the Atlantic salmon catch in that river, but there was no significant relationship between straying rate and water discharge or distance from the river to the River Imsa. The observed straying rate of hatchery Atlantic salmon decreased with increasing number of fish entering the River Imsa. Sexual maturation as parr did not influence the tendency to stray. The results suggest that the establishment of temporary zones, free of fish farms, outside important Atlantic salmon rivers by the fisheries authorities in Norway should be large, whole fjords, to be effective.