Breakpoint analysis: precise localization of genetic markers by means of nonstatistical computation using relatively few genotypes.

Placing new markers on a previously existing genetic map by using conventional methods of multilocus linkage analysis requires that a large number of reference families be genotyped. This paper presents a methodology for placing new markers on existing genetic maps by genotyping only a few individuals in a selected subset of the reference panel. We show that by identifying meiotic breakpoint events within existing genetic maps and genotyping individuals who exhibit these events, along with one nonrecombinant sibling and their parents, we can determine precise location for new markers even within subcentimorgan chromosomal regions. This method also improves detection of errors in genotyping and assists in the observation of chromosome behavior in specific regions.     

Identification and characterization of the familial adenomatous polyposis coli gene.

DNA from 61 unrelated patients with adenomatous polyposis coli (APC) was examined for mutations in three genes (DP1, SRP19, and DP2.5) located within a 100 kb region deleted in two of the patients. The intron-exon boundary sequences were defined for each of these genes, and single-strand conformation polymorphism analysis of exons from DP2.5 identified four mutations specific to APC patients. Each of two aberrant alleles contained a base substitution changing an amino acid to a stop codon in the predicted peptide; the other mutations were small deletions leading to frameshifts. Analysis of DNA from parents of one of these patients showed that his 2 bp deletion is a new mutation; furthermore, the mutation was transmitted to two of his children. These data have established that DP2.5 is the APC gene.     

The European carbon balance. Part 2: croplands

We estimated the long‐term carbon balance [net biome production (NBP)] of European (EU‐25) croplands and its component fluxes, over the last two decades. Net primary production (NPP) estimates, from different data sources ranged between 490 and 846 gC m^?2 yr^?1, and mostly reflect uncertainties in allocation, and in cropland area when using yield statistics. Inventories of soil C change over arable lands may be the most reliable source of information on NBP, but inventories lack full and harmonized coverage of EU‐25. From a compilation of inventories we infer a mean loss of soil C amounting to 17 g m^?2 yr^?1. In addition, three process‐based models, driven by historical climate and evolving agricultural technology, estimate a small sink of 15 g C m^?2 yr^?1 or a small source of 7.6 g C m^?2 yr^?1. Neither the soil C inventory data, nor the process model results support the previous European‐scale NBP estimate by Janssens and colleagues of a large soil C loss of 90 ± 50 gC m^?2 yr^?1. Discrepancy between measured and modeled NBP is caused by erosion which is not inventoried, and the burning of harvest residues which is not modeled. When correcting the inventory NBP for the erosion flux, and the modeled NBP for agricultural fire losses, the discrepancy is reduced, and cropland NBP ranges between ?8.3 ± 13 and ?13 ± 33 g C m^?2 yr^?1 from the mean of the models and inventories, respectively. The mean nitrous oxide (N₂O) flux estimates ranges between 32 and 37 g C Eq m^?2 yr^?1, which nearly doubles the CO₂ losses. European croplands act as small CH₄ sink of 3.3 g C Eq m^?2 yr^?1. Considering ecosystem CO₂, N₂O and CH₄ fluxes provides for the net greenhouse gas balance a net source of 42–47 g C Eq m^?2 yr^?1. Intensifying agriculture in Eastern Europe to the same level Western Europe amounts is expected to result in a near doubling of the N₂O emissions in Eastern Europe. N₂O emissions will then become the main source of concern for the impact of European agriculture on climate.     

Curvularia haloperoxidase: antimicrobial activity and potential application as a surface disinfectant

A presumed antimicrobial enzyme system, the Curvularia haloperoxidase system, was examined with the aim of evaluating its potential as a sanitizing agent. In the presence of hydrogen peroxide, Curvularia haloperoxidase facilitates the oxidation of halides, such as chloride, bromide, and iodide, to antimicrobial compounds. The Curvularia haloperoxidase system caused several-log-unit reductions in counts of bacteria (Pseudomonas spp., Escherichia coli, Serratia marcescens, Aeromonas salmonicida, Shewanella putrefaciens, Staphylococcus epidermidis, and Listeria monocytogenes), yeasts (Candida sp. and Rhodotorula sp.), and filamentous fungi (Aspergillus niger, Aspergillus tubigensis, Aspergillus versicolor, Fusarium oxysporum, Penicillium chrysogenum, and Penicillium paxilli) cultured in suspension. Also, bacteria adhering to the surfaces of contact lenses were killed. The numbers of S. marcescens and S. epidermidis cells adhering to contact lenses were reduced from 4.0 and 4.9 log CFU to 1.2 and 2.7 log CFU, respectively, after treatment with the Curvularia haloperoxidase system. The killing effect of the Curvularia haloperoxidase system was rapid, and 10(6) CFU of E. coli cells/ml were eliminated within 10 min of treatment. Furthermore, the antimicrobial effect was short lived, causing no antibacterial effect against E. coli 10 min after the system was mixed. Bovine serum albumin (1%) and alginate (1%) inhibited the antimicrobial activity of the Curvularia haloperoxidase system, whereas glucose and Tween 20 did not affect its activity. In conclusion, the Curvularia haloperoxidase system is an effective sanitizing system and has the potential for a vast range of applications, for instance, for disinfection of contact lenses or medical devices.     

N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells

The potential of insect cell cultures and larvae infected with recombinant baculoviruses to produce authentic recombinant glycoproteins cloned from mammalian sources was investigated. A comparison was made of the N-linked glycans attached to secreted alkaline phosphatase (SEAP) produced in four species of insect larvae and their derived cell lines plus one additional insect cell line and larvae of one additional species. These data survey N-linked oligosaccharides produced in four families and six genera of the order Lepidoptera. Recombinant SEAP expressed by recombinant isolates of Autographa californica and Bombyx mori nucleopolyhedroviruses was purified from cell culture medium, larval hemolymph or larval homogenates by phosphate affinity chromatography. The N-linked oligosaccharides were released with PNGase-F, labeled with 8- aminonaphthalene-1-3-6-trisulfonic acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by fluorescence imaging. The oligosaccharide structures were confirmed with exoglycosidase digestions. Recombinant SEAP produced in cell lines of Lymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyx mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni , H.virescens , B.mori , and Danaus plexippus contained oligosaccharides that were structurally identical to the 10 oligosaccharides attached to SEAP produced in T.ni cell lines. The oligosaccharide structures were all mannose-terminated. Structures containing two or three mannose residues, with and without core fucosylation, constituted more than 75% of the oligosaccharides from the cell culture and larval samples.      

A Radiation Hybrid Map of the BRCA1 Region

A locus on chromosome 17q, designated “BRCA1,” has been identified as a predisposition gene for breast cancer. A panel of chromosome 17–specific radiation-reduced somatic cell hybrid clones has been assembled for high-resolution mapping of chromosome 17. A series of 35 markers, known to span the BRCA1 locus, were tested against this hybrid panel by PCR assays. Statistical analysis of these data yields a BRCA1 radiation hybrid map at a density sufficient to initiate YAC cloning and pulsed-field gel electrophoretic mapping of the candidate region. In addition, many of the markers reveal genetic polymorphisms and may be tested in breast cancer families and in loss-of-heterozygosity studies of sporadic breast cancers to better define the BRCA1 gene candidate region.     

Haplotypes at LBX1 Have Distinct Inheritance Patterns with Opposite Effects in Adolescent Idiopathic Scoliosis

Adolescent idiopathic scoliosis (AIS) is a clinically significant disorder with high heritability that affects 2–4% of the population. Genome-wide association studies have identified LBX1 as a strong susceptibility locus for AIS in Asian and Caucasian populations. Here we further dissect the genetic association with AIS in a Caucasian population. To identify genetic markers associated with AIS we employed a genome-wide association study (GWAS) design comparing 620 female Caucasian patients who developed idiopathic scoliosis during adolescence with 1,287 ethnically matched females who had normal spinal curves by skeletal maturity. The genomic region around LBX1 was imputed and haplotypes investigated for genetic signals under different inheritance models. The strongest signal was identified upstream of LBX1 (rs11190878, P_trend = 4.18×10^-9, OR = 0.63[0.54–0.74]). None of the remaining SNPs pass the genome-wide significance threshold. We found rs11190870, downstream of LBX1 and previously associated with AIS in Asian populations, to be in modest linkage disequilibrium (LD) with rs11190878 (r² = 0.40, D'' = 0.81). Haplotype analysis shows that rs11190870 and rs11190878 track a single risk factor that resides on the ancestral haplotype and is shared across ethnic groups. We identify six haplotypes at the LBX1 locus including two strongly associated haplotypes; a recessive risk haplotype (TTA, Control_freq = 0.52, P = 1.25×10^-9, OR = 1.56), and a co-dominant protective haplotype (CCG, Control_freq = 0.28, P = 2.75×10^-7, OR = 0.65). Together the association signals from LBX1 explain 1.4% of phenotypic variance. Our results identify two clinically relevant haplotypes in the LBX1-region with opposite effects on AIS risk. The study demonstrates the utility of haplotypes over un-phased SNPs for individualized risk assessment by more strongly delineating individuals at risk for AIS without compromising the effect size.     

Does multiple paternity affect seed mass in angiosperms? An experimental test in Dalechampia scandens

Flowers fertilized by multiple fathers may be expected to produce heavier seeds than those fertilized by a single father. However, the adaptive mechanisms leading to such differences remain unclear, and the evidence inconsistent. Here, we first review the different hypotheses predicting an increase in seed mass when multiple paternity occurs. We show that distinguishing between these hypotheses requires information about average seed mass, but also about within‐fruit variance in seed mass, bias in siring success among pollen donors, and whether siring success and seed mass are correlated. We then report the results of an experiment on Dalechampia scandens (Euphorbiaceae), assessing these critical variables in conjunction with a comparison of seed mass resulting from crosses with single vs. multiple pollen donors. Siring success differed among males when competing for fertilization, but average seed mass was not affected by the number of fathers. Furthermore, paternal identity explained only 3.8% of the variance in seed mass, and siring success was not correlated with the mass of the seeds produced. Finally, within‐infructescence variance in seed mass was not affected by the number of fathers. These results suggest that neither differential allocation nor sibling rivalry has any effect on the average mass of seeds in multiply sired fruits in D. scandens. Overall, the limited paternal effects observed in most studies and the possibility of diversification bet hedging among flowers (but not within flowers), suggest that multiple paternity within fruits or infructescence is unlikely to affect seed mass in a large number of angiosperm species.     

‘Candidatus Competibacter'-lineage genomes retrieved from metagenomes reveal functional metabolic diversity

The glycogen-accumulating organism (GAO) ‘Candidatus Competibacter'' (Competibacter) uses aerobically stored glycogen to enable anaerobic carbon uptake, which is subsequently stored as polyhydroxyalkanoates (PHAs). This biphasic metabolism is key for the Competibacter to survive under the cyclic anaerobic-‘feast'': aerobic-‘famine'' regime of enhanced biological phosphorus removal (EBPR) wastewater treatment systems. As they do not contribute to phosphorus (P) removal, but compete for resources with the polyphosphate-accumulating organisms (PAO), thought responsible for P removal, their proliferation theoretically reduces the EBPR capacity. In this study, two complete genomes from Competibacter were obtained from laboratory-scale enrichment reactors through metagenomics. Phylogenetic analysis identified the two genomes, ‘Candidatus Competibacter denitrificans'' and ‘Candidatus Contendobacter odensis'', as being affiliated with Competibacter-lineage subgroups 1 and 5, respectively. Both have genes for glycogen and PHA cycling and for the metabolism of volatile fatty acids. Marked differences were found in their potential for the Embden–Meyerhof–Parnas and Entner–Doudoroff glycolytic pathways, as well as for denitrification, nitrogen fixation, fermentation, trehalose synthesis and utilisation of glucose and lactate. Genetic comparison of P metabolism pathways with sequenced PAOs revealed the absence of the Pit phosphate transporter in the Competibacter-lineage genomes—identifying a key metabolic difference with the PAO physiology. These genomes are the first from any GAO organism and provide new insights into the complex interaction and niche competition between PAOs and GAOs in EBPR systems.