Novel blue light-sensitive proteins from a metagenomic approach

A microarray‐based approach was used to screen a soil metagenome for the presence of blue light (BL) photoreceptor‐encoding genes. The microarray carried 149 different 54‐mer oligonucleotides, derived from consensus sequences of light, oxygen and voltage (LOV) domain BL photoreceptor genes. Calibration of the microarrays allowed the detection of minimally 50 ng of genomic DNA against a background of 2–5 μg of genomic DNA. Identification of a positive cosmid clone was still possible for an amount of 0.25 ng against a background of 10 μg of labelled DNA clones. The array could readily identify targets carrying 4% sequence mismatch. Using the LOV microarray, up to 1200 library clones in concentrations of c. 20 ng each with a c. 40 kb insert size could be screened in a single batch. After calibration and reliability controls, the microarray was probed with cosmid‐cloned DNA from the thermophilic fraction of a soil sample. From this approach, a novel gene was isolated that encodes a protein consisting of several Per‐Arnt‐Sim domains, a LOV domain associated to a histidine kinase and a response regulator domain. The novel gene showed highest similarity to a known sequence from Kineococcus radiotolerans SRS30216 (58% identity for the LOV domain only) and to a gene from Methylibium petroleiphilum PM1 (57% identity). The gene, designated as ht‐met1 (Hamburg Thermophile Metagenome 1), was isolated and fully sequenced (3615 bp). ht‐met1 is followed by a second open reading frame encoding a Fe‐chelatase, an arrangement quite frequent for BL photoreceptors. The LOV domain region of ht‐met1 was subcloned and expressed yielding a fully functional, flavin‐containing LOV domain. Irradiation generated the typical LOV photochemistry, with the transient formation of a flavin‐protein photoadduct. The dark recovery lifetime was found as τ_REC = 120 s (20°C) and is among the fastest ones determined so far for bacterial LOV domains.     

Molecular characterization of a trafficking organelle: dissecting the axonal paths of calsyntenin-1 transport vesicles

Kinesin motors play crucial roles in the delivery of membranous cargo to its destination and thus for the establishment and maintenance of cellular polarization. Recently, calsyntenin-1 was identified as a cargo-docking protein for Kinesin-1-mediated axonal transport of tubulovesicular organelles along axons of central nervous system neurons. To further define the function of calsyntenin-1, we immunoisolated calsyntenin-1 organelles from murine brain homogenates and determined their proteome by MS. We found that calsyntenin-1 organelles are endowed with components of the endosomal trafficking machinery and contained the β-amyloid precursor protein (APP). Detailed biochemical analyses of calsyntenin-1 immunoisolates in conjunction with immunocytochemical colocalization studies with cultured hippocampal neurons, using endosomal marker proteins for distinct subcompartments of the endosomal pathways, indicated that neuronal axons contain at least two distinct, nonoverlapping calsyntenin-1-containing transport packages: one characterized as early-endosomal, APP positive, the other as recycling-endosomal, APP negative. We postulate that calsyntenin-1 acts as a general mediator of anterograde axonal transportation of endosomal vesicles. In this role, calsyntenin-1 may actively contribute to axonal growth and pathfinding in the developing as well as to the maintenance of neuronal polarity in the adult nervous system; further, it may actively contribute to the stabilization of APP during its anterograde axonal trajectory.     

A Community-Based Study of Factors Associated with Continuing Transmission of Lymphatic Filariasis in Leogane,Haiti

Seven rounds of mass drug administration (MDA) have been administered in Leogane, Haiti, an area hyperendemic for lymphatic filariasis (LF). Sentinel site surveys showed that the prevalence of microfilaremia was reduced to <1% from levels as high as 15.5%, suggesting that transmission had been reduced. A separate 30-cluster survey of 2- to 4-year-old children was conducted to determine if MDA interrupted transmission. Antigen and antifilarial antibody prevalence were 14.3% and 19.7%, respectively. Follow-up surveys were done in 6 villages, including those selected for the cluster survey, to assess risk factors related to continued LF transmission and to pinpoint hotspots of transmission. One hundred houses were mapped in each village using GPS-enabled PDAs, and then 30 houses and 10 alternates were chosen for testing. All individuals in selected houses were asked to participate in a short survey about participation in MDA, history of residence in Leogane and general knowledge of LF. Survey teams returned to the houses at night to collect blood for antigen testing, microfilaremia and Bm14 antibody testing and collected mosquitoes from these communities in parallel. Antigen prevalence was highly variable among the 6 villages, with the highest being 38.2% (Dampus) and the lowest being 2.9% (Corail Lemaire); overall antigen prevalence was 18.5%. Initial cluster surveys of 2- to 4-year-old children were not related to community antigen prevalence. Nearest neighbor analysis found evidence of clustering of infection suggesting that LF infection was focal in distribution. Antigen prevalence among individuals who were systematically noncompliant with the MDAs, i.e. they had never participated, was significantly higher than among compliant individuals (p<0.05). A logistic regression model found that of the factors examined for association with infection, only noncompliance was significantly associated with infection. Thus, continuing transmission of LF seems to be linked to rates of systematic noncompliance.     

Neural induction. A bird's eye view

Since the discovery of the phenomenon of neural induction by Spemann and Mangold in 1924, considerable effort has been invested in identifying the signals produced by the organizer that are responsible for diverting the fate of cells from epidermal to neural. Substantial progress has been made only recently by the finding in amphibians that BMP4 is a neural inhibitor and epidermal inducer, and that endogenous antagonists of BMPs are secreted by the organizer. However, recent results in the chick point to the existence of other, upstream events required before BMP inhibition stabilizes neural fates. Here we take a critical view of the evidence for and against the view that BMP inhibition is a sufficient trigger for neural induction in different vertebrates.     

Isolation of unique nucleic acid sequences from rhizobia by genomic subtraction: Applications in microbial ecology and symbiotic gene analysis

A subtraction hybridization and PCR amplification procedure was used to isolate two Rhizobium DNA probes which exhibited high degrees of specificity at different levels of taxonomic organization and which could be used as tools for detection of rhizobia in ecological studies. First, a probe was isolated from Rhizobium leguminosarum bv. trifolii strain P3 by removing those Sau3A restriction fragments from a P3 DNA digest which cross hybridized with pooled DNA from seven other strains of the same biovar. The remaining restriction fragments hybridized to DNA from strain P3 but not to DNA from any of the seven other strains. In a similar experiment another DNA probe, specific for the Rhizobium leguminosarum bv. phaseoli and Rhizobium tropici group, was generated by removing sequences from R. leguminosarum bv phaseoli strain Kim 5s with pooled subtracter DNA from eight other Rhizobium, Bradyrhizobium and Agrobacterium species. The same subtraction hybridization technique was also used to isolate symbiotic genes from a Rhizobium species. Results from a 1:1 subtractive DNA hybridization of the broad host range Rhizobium sp NGR234 against highly homologous S. fredii USDA257, combined with those from competitive RNA hybridizations to cosmid digests of the NGR234 symbiotic plasmid, allowed the identification of several NGR234 loci which were flavonoid-inducible and not present in S. fredii USDA257. One of these, ORF-1, was highly homologous to the leucine responsive regulatory protein of E. coli.     

A note on the nutrition of Stylaria lacustris (Oligochaeta: Naididae)

Stylaria lacustris (L.) was offered ¹⁴C-labelled algae. The ingestion rate of diatoms was 0.79 g carbon/hour at 19°C. Diatoms (Nitzschia actinastroides) were well assimilated, green algae (Scenedesmus acuminatus) were practically not assimilated. The less assimilable algae are ingested at higher rates, indicating a regulation mechanism. Biomass doubling time was estimated to be about 3.6 days at 19°C.     

Energy flow in four different field populations of Ancylus fluviatilis (Gastropoda-Basommatophora)

Summary Four brook-dwelling populations of Ancylus fluviatilis were studied. Data on life-cycle, specimen number, biomass and egg capsule biomass are reported. By means of experimental data on ingestion, assimilation, growth and egg-production, energy balances are calculated for whole populations.Mean annual biomass turned out to be 26.58–64.88 mg C/m². Mean annual biomass turn-over ratio (production/biomass) was 2.64–5.95. Cohort biomass turnover ratio was 4.11–6.09 for the whole population. Young specimens show considerably higher production efficiencies than adult ones. Production by the smallest size classes is a very considerable part of whole population production. Net production efficiency of the whole population was 11.2–11.8%, gross production efficiency 5.6–6.5%. An important factor for variation between different populations is temperature, but other factors, such as food supply, may be important, too. Experimental production estimates were compaerd with the Allen curve method and the instantaneous growth method.Differences due to different methods of production measurement are discussed. The range of values for different efficiencies lies within the range reported for other freshwater snails. Interpopulation variation in turnover ratios nearly equals variation reported for different species of univoltine snails.Supported by the Deutsche Forschungsgemeinschaft.