Head Proteins from T-Even Bacteriophages II. Physical and Chemical Characterization

Three classes of structural head proteins, having molecular weights of 43,000, 18,000, and 11,000 daltons, were isolated from the T-even bacteriophages and were characterized. Based on electrophoretic studies, the 43,000-dalton class contained one major protein and one (or two) minor components, the 18,000-dalton class contained two protein components, and the 11,000-dalton class contained one major component. The N-terminal residues for the 43,000- and the 11,000-dalton classes were alanine, and the N-terminal residues for the 18,000-dalton class were methionine and alanine. Of the three classes of proteins, the 18,000-dalton proteins were the most acidic, whereas the 11,000-dalton proteins were the most basic. The amino acid composition of the 11,000-dalton class revealed that methionine and cysteine were absent and lysine, histidine, and tryptophan content was higher in the 11,000-dalton class than in the other two classes of proteins. Estimates of the relative number of the three classes of structural proteins were made and indicated that there were between 1,600 and 2,000 subunits of the 43,000-dalton proteins, 100 to 200 of the 18,000-dalton proteins, and 1,000 to 1,500 of the 11,000-dalton proteins. Evidence was presented that the 43,000-dalton proteins and the 11,000-dalton proteins readily formed aggregates with themselves but not with each other. The significance of these interactions to the structure of the T-even phage head was discussed.     

Competition for hydrogen between sulphate-reducing bacteria and methanogenic bacteria from the human large intestine

Sulphate-reducing activity in human faecal slurries was followed by measuring sulphide production. Sulphate-reducing bacteria (SRB) were found to outcompete methanogenic bacteria (MB) for the mutual substrate hydrogen in faecal slurries from methane- and non-methane-producing individuals mixed together. When molybdate (20 mmol/l) was added to these slurries, sulphate reduction was inhibited and methanogenesis became the major route of electron disposal. Sulphide production was stimulated by the addition of 20 mmol/l sulphate in non-methanogenic but not in methanogenic slurries. In methanogenic slurries that contained the methanogen inhibitor 2-bromoethanesulphonic acid (BES), hydrogen accumulated whilst sulphide levels were unaffected, confirming the absence of SRB in methanogenic faeces. The addition of nitrate (10 mmol/l) to faecal slurries completely inhibited methanogenesis but only slightly reduced sulphate reduction. The sulphated mucopolysaccharides, chondroitin sulphate and mucin, strongly stimulated sulphide production in non-methanogenic faecal slurries only, suggesting that these substances may be a potential source of sulphate in the large gut.     

Anatomy of mitochondrial DNA from Paramecium aurelia

Summary The linear genome of mitochondrial DNA from four species of Paramecium aurelia was investigated with respect to restriction endonuclease fragments, location and number of ribosomal RNA genes, and interspecies EcoRI and HindIII fragment homologies. One copy of each of the rRNA genes was found in all four species and the 14s and 20s rRNA genes were separated by at least 3,000 bp. R-Loop analysis of the 20s rRNA gene did not reveal the presence of an intervening sequence. Interspecies homology studies showed species 1, 5, and 7 to have a high degree of homology but species 4 was less than 50% homologous to species 1 mt DNA. For all four species, rRNA genes showed good homology indicating that these DNA sequences are highly conserved, even between species having many non-homologous regions. A major region of DNA which displayed little homology between species 1 and 4 was that fragment containing sequences essential for initiation of DNA replication.     

Solutions for PCR,cloning and sequencing errors in population genetic analysis

PCR and sequencing artefacts can seriously bias population genetic analyses, particularly of populations with low genetic variation such as endangered vertebrate populations. Here, we estimate the error rates, discuss their population genetics implications, and propose a simple detection method that helps to reduce the risk of accepting such errors. We study the major histocompatibility complex (MHC) class IIB of guppies, Poecilia reticulata and find that PCR base misincorporations inflate the apparent sequence diversity. When analysing neutral genes, such bias can inflate estimates of effective population size. Previously suggested protocols for identifying genuine alleles are unlikely to exclude all sequencing errors, or they ignore genuine sequence diversity. We present a novel and statistically robust method that reduces the likelihood of accepting PCR artefacts as genuine alleles, and which minimises the necessity of repeated genotyping. Our method identifies sequences that are unlikely to be a PCR artefact, and which need to be independently confirmed through additional PCR of the same template DNA. The proposed methods are recommended particularly for population genetic studies that involve multi-template DNA and in studies on genes with low genetic diversity.     

A novel alpha1,2-fucosyltransferase (CE2FT-2) in Caenorhabditis elegans generates H-type 3 glycan structures

The 1,2-fucosyltransferase family (1,2FT) is the largest familyof glycosyltransferases in the genome of the free-living nematodeCaenorhabditis elegans, and early evidence suggests that eachmember may have a unique activity. Here we describe a C. elegansgene (designated CE2FT-2) encoding an 1,2FT that has the potentialto generate the sequence Fuc1-2Galβ1-3GalNAc-R, which isthe H-type 3 blood group structure. The CE2FT-2 cDNA encodesa putative transmembrane protein that shows 42% amino acid identityto a previously cloned C. elegans 1,2FT (termed CE2FT-1), buthas a very low identity (16–20%) to 1,2FT sequences inhumans, rabbits, and mice. A recombinant form of CE2FT-2 expressedin human 293T cells has a high 1,2FT activity toward Galβ1-3GalNAc-O-pNP,but unexpectedly, the enzyme is inactive toward the acceptorGalβ-O-phenyl. Thus, CE2FT-2 differs from all other 1,2FTspreviously described from animals that all utilize Galβ-O-phenyl.CE2FT-2 is expressed at all stages of worm development, butremarkably, promoter analysis of the CE2FT-2 gene using greenfluorescent protein reporter constructs indicates that the CE2FT-2is expressed exclusively in pharyngeal cells of the worm fromembryo to an adult stage. Because pharyngeal cells are knownto secrete their glycoconjugates to the nematode surface, theseresults may indicate that products of CE2FT-2 contribute tointeractions of the nematode with its environment or are usedas ligands for bacterial attachment. These findings, along withthose on other 1,2FTs in C. elegans, suggest that each 1,2FTin this organism may have a unique acceptor specificity, expressionpattern, and biological function.     

Regulation of proximal-distal intercalation during limb regeneration in the axolotl (Ambystoma mexicanum)

Intercalation is the process whereby cells located at the boundary of a wound interact to stimulate proliferation and the restoration of the structures between the boundaries that were lost during wounding. Thus, intercalation is widely considered to be the mechanism of regeneration. When a salamander limb is amputated, the entire cascade of regeneration events is activated, and the missing limb segments and their boundaries (joints) as well as the structures within each segment are regenerated. Therefore, in an amputated limb it is not possible to distinguish between intersegmental regeneration (formation of new segments/joints) and intrasegmental regeneration (formation of structures within a given segment), and it is not possible to study the differential regulation of these two processes. We have used two models for regeneration that allow us to study these two processes independently, and report that inter- and intrasegmental regeneration are different processes regulated by different signaling pathways. New limb segments/joints can be regenerated from cells that dedifferentiate to form blastema cells in response to signaling that is mediated in part by fibroblast growth factor.     

Expert Panel on Weight Loss Surgery: Executive Report Update

Rapid shifts in the demographics and techniques of weight loss surgery (WLS) have led to new issues, new data, new concerns, and new challenges. In 2004, this journal published comprehensive evidence‐based guidelines on WLS. In this issue, we've updated those guidelines to assure patient safety in this fast‐changing field. WLS involves a uniquely vulnerable population in need of specialized resources and ongoing multidisciplinary care. Timely best‐practice updates are required to identify new risks, develop strategies to address them, and optimize treatment. Findings in these reports are based on a comprehensive review of the most current literature on WLS; they directly link patient safety to methods for setting evidence‐based guidelines developed from peer‐reviewed scientific publications. Among other outcomes, these reports show that WLS reduces chronic disease risk factors, improves health, and confers a survival benefit on those who undergo it. The literature also shows that laparoscopy has displaced open surgery as the predominant approach; that government agencies and insurers only reimburse procedures performed at accredited WLS centers; that best practice care requires close collaboration between members of a multidisciplinary team; and that new and existing facilities require wide‐ranging changes to accommodate growing numbers of severely obese patients. More than 100 specialists from across the state of Massachusetts and across the many disciplines involved in WLS came together to develop these new standards. We expect them to have far‐reaching effects of the development of health care policy and the practice of WLS.     

Geographic differentiation in the house fly estimated by microsatellite and mitochondrial variation

Gene flow over very large geographic scales has been investigated in few species. Examples include Drosophila melanogaster, Drosophila subobscura, Drosophila simulans, and the Mediterranean fruit fly (Ceratitis capitata). The cosmopolitan house fly, a highly vagile, fecund, colonizing species offers an additional exemplar. Genotypes at seven microsatellite loci were scored in 14 widely separated natural house fly populations from the Nearctic, neotropics, Afrotropics, Palearctic, and Asia. Allelic diversities and heterozygosities differed significantly among populations. Averaged over all populations, Weir and Cockerham's theta = 0.13 and RST = 0.20. Pairwise genetic distance measures were uncorrelated with geographic distance. Microsatellite frequencies were compared with mitochondrial data from 13 of the same populations in which theta = 0.35 and Nei's GST = 0.72. Mitochondrial variation indicated up to threefold greater indices of genetic differentiation than the microsatellites. We were unable to draw any biogeographical inferences from these results or from tree or network topologies constructed from the genetic data. It is likely that high microsatellite diversities, mutation rates, and homoplasy greatly compromised their usefulness in estimating gene flow. House fly colonization dynamics include a large number of primary and secondary colonizations coupled with substantial genetic drift, but no detectable bottlenecks.     

Impacts of Organic and Conventional Crop Management on Diversity and Activity of Free-Living Nitrogen Fixing Bacteria and Total Bacteria Are Subsidiary to Temporal Effects

A three year field study (2007–2009) of the diversity and numbers of the total and metabolically active free-living diazotophic bacteria and total bacterial communities in organic and conventionally managed agricultural soil was conducted using the Nafferton Factorial Systems Comparison (NFSC) study, in northeast England. Fertility management appeared to have little impact on both diazotrophic and total bacterial communities. However, copy numbers of the nifH gene did appear to be negatively impacted by conventional crop protection measures across all years suggesting diazotrophs may be particularly sensitive to pesticides. Impacts of crop management were greatly overshadowed by the influence of temporal effects with diazotrophic communities changing on a year by year basis and from season to season. Quantitative analyses using qPCR of each community indicated that metabolically active diazotrophs were highest in year 1 but the population significantly declined in year 2 before recovering somewhat in the final year. The total bacterial population in contrast increased significantly each year. It appeared that the dominant drivers of qualitative and quantitative changes in both communities were annual and seasonal effects. Moreover, regression analyses showed activity of both communities was significantly affected by soil temperature and climatic conditions.