Opportunities for natural selection on DNA and protein at the Adh locus in Drosophila melanogaster

A study was made of environmental and genetic factors affecting the quantity and disposition of the alcohol dehydrogenase (ADH) protein in Drosophila melanogaster. It was found that the amount of enzyme per fly is greatly influenced by the environmental conditions in which it develops. A critical factor is the concentration of yeast in the medium. A high concentration of yeast can double the quantity of ADH. The yeast appears to act through the provision of protein, and the protein to act through the provision of threonine, which is already known to induce ADH in fungi. Various genetic factors affect the quantity of enzyme. Males have more ADH than females. Files homozygous for the Fast allele have more ADH than those homozygous for the slow allele, and the difference is greater in females than in males. One particular line (ve), homozygous for Slow, has approximately half the normal quantity of enzyme, and the quantity segregates with the electrophoretic allele. Lines differ in the relative amounts of ADH in the gut (including Malpighian tubules) and the fat body. In general it seems that slow lines have relatively more enzyme in the fat body. In a cross between ve and a line homozygous to Fast, the difference in tissue distribution segregated with the electrophoretic allele. It is argued, but not demonstrated, that the differences in quantity and tissue distribution are due to nucleotide substitutions in noncoding regions close to, or within, the structural gene. It seems likely that the observed environmental and genetic differences in the quantity and disposition of ADH will influence the relative selective values of the electrophoretic genotypes.     

Bioaccumulation of marine pollutants.

Bioaccumulation of pollutants can occur from sea water, from suspended particles, from sediments and through food chains. The rate at which accumulation occurs in an organism depends not only on the availability of the pollutant but also on a whole range of biological, chemical and environmental factors. The ultimate level which is reached is governed by the ability of the organism to excrete the pollutant or, alternatively, store it. This latter course often leads to the attainment of very high concentrations and sometimes no equilibrium level is ever reached. Two particular topics which are considered are the biological amplification of pollutants along food chains and the development of tolerance which sometimes occurs.     

Antibody-coated bacteria in the urine of preschool and school-aged girls with asymptomatic bacteriuria.

Urine samples from 3564 girls aged 2 to 13 years were screened for evidence of infection. Cultures were positive (bacteria count, more than 10(5)/ml) in 61 (1.7%) by the dipslide method and in 55 (1.5%) by standard culture techniques. In 13 (23.6%) of the 55, antibody-coated bacteria (ACB) were detected in the urine. The clinical, bacteriologic, radiologic and urinalysis findings in children with ACB were no different from those in children in whom the bacteria were not coated. Direct examination of uncentrifuged urine under high power revealed one or more bacteria per two high-power fields in 96% of infected urine samples and in only 7% of noninfected samples. Five or more leukocytes per high-power field in centrifuged urine were detected in 36.7% of infected urine samples but not in noninfected samples. The ACB test did not differentiate between asymptomatic bacteriuria with parenchymal scarring or vesicoureteral reflux or both and asymptomatic bacteriuria without these abnormalities.     

Centrosomal enrichment and proteasomal degradation of SYS-1/β-catenin requires the microtubule motor dynein

The Caenorhabditis elegans Wnt/β-catenin asymmetry (WβA) pathway utilizes asymmetric regulation of SYS-1/β-catenin and POP-1/TCF coactivators. WβA differentially regulates gene expression during cell fate decisions, specifically by asymmetric localization of determinants in mother cells to produce daughters biased toward their appropriate cell fate. Despite the induction of asymmetry, β-catenin localizes symmetrically to mitotic centrosomes in both mammals and C. elegans. Owing to the mitosis-specific localization of SYS-1 to centrosomes and enrichment of SYS-1 at kinetochore microtubules when SYS-1 centrosomal loading is disrupted, we investigated active trafficking in SYS-1 centrosomal localization. Here, we demonstrate that trafficking by microtubule motor dynein is required to maintain SYS-1 centrosomal enrichment, by dynein RNA interference (RNAi)-mediated decreases in SYS-1 centrosomal enrichment and by temperature-sensitive allele of the dynein heavy chain. Conversely, we observe depletion of microtubules by nocodazole treatment or RNAi of dynein-proteasome adapter ECPS-1 exhibits increased centrosomal enrichment of SYS-1. Moreover, disruptions to SYS-1 or negative regulator microtubule trafficking are sufficient to significantly exacerbate SYS-1 dependent cell fate misspecifications. We propose a model whereby retrograde microtubule-mediated trafficking enables SYS-1 enrichment at centrosomes, enhancing its eventual proteasomal degradation. These studies support the link between centrosomal localization and enhancement of proteasomal degradation, particularly for proteins not generally considered “centrosomal.”     

Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells

NK cells utilize a large array of receptors to screen their surroundings for aberrant or virus‐infected cells. Given the vast diversity of receptors expressed on NK cells we seek to identify receptors involved in the recognition of HIV‐1‐infected cells. By combining an unbiased large‐scale screening approach with a functional assay, we identify TRAIL to be associated with NK cell degranulation against HIV‐1‐infected target cells. Further investigating the underlying mechanisms, we demonstrate that TRAIL is able to elicit multiple effector functions in human NK cells independent of receptor‐mediated induction of apoptosis. Direct engagement of TRAIL not only results in degranulation but also IFNγ production. Moreover, TRAIL‐mediated NK cell activation is not limited to its cognate death receptors but also decoy receptor I, adding a new perspective to the perceived regulatory role of decoy receptors in TRAIL‐mediated cytotoxicity. Based on these findings, we propose that TRAIL not only contributes to the anti‐HIV‐1 activity of NK cells but also possesses a multifunctional role beyond receptor‐mediated induction of apoptosis, acting as a regulator for the induction of different effector functions.     

In vitro eradication of abasic site-mediated DNA–peptide/protein cross-links by Escherichia coli long-patch base excision repair

Apurinic/apyrimidinic (AP or abasic) sites are among the most abundant DNA lesions. Numerous proteins within different organisms ranging from bacteria to human have been demonstrated to react with AP sites to form covalent Schiff base DNA–protein cross-links (DPCs). These DPCs are unstable due to their spontaneous hydrolysis, but the half-lives of these cross-links can be as long as several hours. Such long-lived DPCs are extremely toxic due to their large sizes, which physically block DNA replication. Therefore, these adducts must be promptly eradicated to maintain genome integrity. Herein, we used in vitro reconstitution experiments with chemically synthesized, stable, and site-specific Schiff base AP-peptide/protein cross-link analogs to demonstrate for the first time that this type of DPC can be repaired by Escherichia coli (E. coli) long-patch base excision repair. We demonstrated that the repair process requires a minimum of three enzymes and five consecutive steps, including: (1) 5′-DNA strand incision of the DPC by endonuclease IV; (2 to 4) strand-displacement DNA synthesis, removal of the 5′-deoxyribose phosphate-peptide/protein adduct-containing flap, and gap-filling DNA synthesis by DNA polymerase I; and (5) strand ligation by a ligase. We further demonstrated that endonuclease IV plays a major role in incising an AP-peptide cross-link within E. coli cell extracts. We also report that eradicating model AP-protein (11.2–36.1 kDa) DPCs is less efficient than that of an AP-peptide_10mer cross-link, supporting the emerging model that proteolysis is likely required for efficient DPC repair.     

A mechanism of abiotic immobilization of nitrate in forest ecosystems: the ferrous wheel hypothesis

Forest soils, rather than woody biomass, are the dominant long‐term sink for N in forest fertilization studies and, by inference, for N from atmospheric deposition. Recent evidence of significant abiotic immobilization of inorganic‐N in forest humus layers challenges a previously widely held view that microbial processes are the dominant pathways for N immobilization in soil. Understanding the plant, microbial, and abiotic mechanisms of N immobilization in forest soils has important implications for understanding current and future carbon budgets. Abiotic immobilization of nitrate is particularly perplexing because the thermodynamics of nitrate reduction in soils are not generally favorable under oxic conditions. Here we present preliminary evidence for a testable hypothesis that explains abiotic immobilization of nitrate in forest soils. Because iron (and perhaps manganese) plays a key role as a catalyst, with Fe(II) reducing nitrate and reduced forms of carbon then regenerating Fe(II), we call this ‘the ferrous wheel hypothesis’. After nitrate is reduced to nitrite, we hypothesize that nitrite reacts with dissolved organic matter through nitration and nitrosation of aromatic ring structures, thus producing dissolved organic nitrogen (DON). In addition to ignorance about mechanisms of DON production, little is known about DON dynamics in soil and its fate within ecosystems. Evidence from leaching and watershed studies suggests that DON production and consumption may be largely uncoupled from seasonal biological processes, although biological processes ultimately produce the DOC and reducing power that affect DON formation and the entire N cycle. The ferrous wheel hypothesis includes both biological and abiological processes, but the reducing power of plant‐derived organic matter may build up over seasons and years while the abiotic reduction of nitrate and reaction of organic matter with nitrite may occur in a matter of seconds after nitrate enters the soil solution.     

Physical mapping across an avirulence locus of Phytophthora infestans using a highly representative, large-insert bacterial artificial chromosome library

The oomycete plant pathogen Phytophthora infestans is the causal agent of late blight, one of the most devastating diseases of potato worldwide. As part of efforts to clone avirulence (Avr) genes and pathogenicity factors from P. infestans, we have constructed a bacterial artificial chromosome (BAC) library from an isolate containing six Avr genes. The BAC library comprises clones with an average insert size of 98 kb and represents an estimated 10 genome equivalents. A three-dimensional pooling strategy was developed to screen the BAC library for amplified fragment length polymorphism (AFLP) markers, as this type of marker has been extensively used in construction of a P. infestans genetic map. Multiple positive clones were identified for each AFLP marker tested. The pools were used to construct a contig of 11 BAC clones in a region of the P. infestans genome containing a cluster of three avirulence genes. The BAC contig is predicted to encompass the Avr11 locus but mapping of the BAC ends will be required to determine if the Avr3 and Avr10 loci are also present in the BAC contig. These results are an important step towards the positional cloning of avirulence genes from P. infestans, and the BAC library represents a valuable resource for largescale studies of oomycete genome organisation and gene content.