Bacterial transformation: ComFA is a DNA‐dependent ATPase that forms complexes with ComFC and DprA

Pneumococcal natural transformation contributes to genomic plasticity, antibiotic resistance development and vaccine escape. Streptococcus pneumoniae, like many other naturally transformable species, has evolved sophisticated protein machinery for the binding and uptake of DNA. Two proteins encoded by the comF operon, ComFA and ComFC, are involved in transformation but their exact molecular roles remain unknown. In this study, we provide experimental evidence that ComFA binds to single stranded DNA (ssDNA) and has ssDNA‐dependent ATPase activity. We show that both ComFA and ComFC are essential for the transformation process in pneumococci. Moreover, we show that these proteins interact with each other and with other proteins involved in homologous recombination, such as DprA, thus placing the ComFA‐ComFC duo at the interface between DNA uptake and DNA recombination during transformation.     

Reconstructing the plant mitochondrial genome for marker discovery: a case study using Pinus

Whole‐genome‐shotgun (WGS) sequencing of total genomic DNA was used to recover ~1 Mbp of novel mitochondrial (mtDNA) sequence from Pinus sylvestris (L.) and three members of the closely related Pinus mugo species complex. DNA was extracted from megagametophyte tissue from six mother trees from locations across Europe, and 100‐bp paired‐end sequencing was performed on the Illumina HiSeq platform. Candidate mtDNA sequences were identified by their size and coverage characteristics, and by comparison with published plant mitochondrial genomes. Novel variants were identified, and primers targeting these loci were trialled on a set of 28 individuals from across Europe. In total, 31 SNP loci were successfully resequenced, characterizing 15 unique haplotypes. This approach offers a cost‐effective means of developing marker resources for mitochondrial genomes in other plant species where reference sequences are unavailable.     

Genetic analysis of variant pilin genes from Neisseria gonorrhoeae P9 cloned in Escherichia coli: physical and immunological properties of encoded pilins

A series of genomic DNA fragments that encode gonococcal pilins from four well-characterized pilus variants of Neisseria gonorrhoeae strain P9 have been cloned in Escherichia coli K12. At least nine classes of cloned P9 pilin genes have been identified on the basis of restriction mapping of cloned pilin-encoding DNA and physical and immunological analysis of expressed pilin proteins. Each antigenic variant of strain P9 possesses many genomic segments of pilin gene information, although our results suggest that strain P9 contains only a single pilin-expressing (pilE) locus.     

Mutation detection in the breast cancer gene BRCA1 using the protein truncation test

About 400 distinct mutations have been defined in the BRCA1 gene, and these are spread fairly evenly through the 5592 bp of coding DNA. This circumstance presents a formidable challenge for mutation screening. Apart from total direct sequencing, the preferred screening method has been single-strand conformation polymorphism (SSCP) gels, with a smaller input from constant denaturant gradient electrophoresis (CDGE), heteroduplex (HD) analysis, and mismatch cleavage. The protein truncation test (PTT) was used early in BRCA1 mutation screening but has not been widely adopted, perhaps because a straightforward analysis of the whole BRCA1 gene requires working with RNA and all its perceived problems. The present work was undertaken to assess the practicality of using the PTT under routine conditions for the screening of long genes such as BRCA1 that are not highly expressed in lymphocytes. We conclude that, provided RNA preparation is carried out effectively and consistently, the PTT approach has significant advantages over other methodologies such as SSCP gels.     

Immunofluorescent Assay for the Marine Ammonium-Oxidizing Bacterium Nitrosococcus oceanus

Nitrification is one of the important microbiological transformations of nitrogen in the ocean. Traditional enrichment-culture methods for enumerating the autotrophic bacteria which oxidize ammonium to nitrite are very time consuming (months) and are believed to seriously underestimate natural abundances. A fluorescent-antibody assay for a marine ammonium-oxidizing bacterium was developed to provide a rapid and direct means of identifying these microorganisms. Antibodies to Nitrosococcus oceanus were prepared and tested against pure cultures of marine, freshwater, and soil ammonium oxidizers and against bacteria from natural seawater samples. Cell counts of culture samples determined by the fluorescent-antibody assay agreed with hemacytometer and acridine orange counts. Our results demonstrated that the immunofluorescent assay is a powerful tool for the detection of Nitrosococcus in the marine environment.     

On the Identification of α- and β-Tubulin Subunits

Abstract: Confusion appears to have arisen in the literature regarding the designation of α-and β-tubulin in polyacrylamide gels. The presence or absence of 8 M-urea in sodium dodecyl sulfate (SDS) polyacrylamide gels leads to different patterns for unalkylated tubulin subunits (and other proteins), making difficult the designation of the α and β subunits by original definition using electrophoretic mobility in the molecular weight dimension. The specific biochemical property of posttranslational tyrosylation of the α subunit has been used to identify further this subunit. Under all conditions tested, the β subunit has been found to be more acidic than the α subunit, with isoelectric point differences that agree with theoretical and published values. If the tubulin subunits are reduced and alkylated, the β subunit migrates more rapidly in SDS polyacrylamide gels, with or without urea present. However, unalkylated tubulin subunits can comigrate or even reverse their relative mobility if 8 M-urea-SDS polyacrylamide gels are used for subunit separation. The results also confirm the earlier reports that the post-translational tyrosylation of protein appears exclusively restricted to α-tubulin and can be demonstrated in an in vivo situation. In addition, the results suggest that only the α₂ subunit of tubulin is tyrosylated.     

East Atlantic teleconnection pattern and the decline of a common arctiid moth

Teleconnection patterns are large‐scale atmospheric circulation systems and variation in them is often associated with impacts on climate and weather over broad areas. Arctia caja L. is a well‐known, widespread and charismatic tiger moth. In recent decades, the abundance of A. caja in UK has fallen abruptly. The annual abundance of A. caja in UK is known to be affected adversely by wet winter weather and warm spring temperatures. We examined A. caja population dynamics from 1968 to 1999 for weather and climatological effects. Population growth rate displayed endogenous effects of abundance in the previous two seasons. Accounting for this, growth rate in the present season was still affected significantly by winter precipitation and spring temperature. Annual abundance of A. caja was inversely related to winter East Atlantic teleconnection pattern (winter EA index) and annual population growth rate was inversely related to winter EA in the present and previous two seasons. An index of the North Atlantic Oscillation (NAO), commonly used as an indicator of winter climate in northern Europe, did not show a significant relationship with growth rate. We noted, for the first time, that the winter EA index has increased steadily over the past five decades. The model presented here therefore implies a further decline of A. caja population growth rates and abundance in the future. This is the first demonstration of a relationship between EA and population dynamics and indicates the EA and other lesser‐known teleconnection patterns may prove useful in modeling the ecological effects of climate change.