Trichoptera (Insecta) from new Guinea∗

This paper is a systematic report on a collection of caddisflies from Papua‐New Guinea. Twenty‐four taxa are identified to species and nine others are determined only to the generic level. Te new species (Diplectrona papuana n. sp., D. subtriangulaia n. sp., Herbertorossia kimminsi n. sp., Hydropsyche papuana n. sp., Cheumatopsyche beroni n. sp., Polycentropus mounthageni n. sp., P. elegans n. sp., Polyplectropus chapmani n. sp., Oecetis kimminsi n. sp. and Triaenodes lelefominicus n.sp.)and the females of Polycentropus similis Kimm, and Polycentropus sinuosus Kimm, are described. New faunistic data is presented for the other species which is supplemented in a few cases by taxonomic notes. The genera Cheumatopsyche, Polyplectropus and Nyctiophylax are new to the fauna of New Guinea.     

Differential requirement of DNA replication factors for subtelomeric ARS consensus sequence protosilencers in Saccharomyces cerevisiae

The establishment of silent chromatin requires passage through S-phase, but not DNA replication per se. Nevertheless, many proteins that affect silencing are bona fide DNA replication factors. It is not clear if mutations in these replication factors affect silencing directly or indirectly via deregulation of S-phase or DNA replication. Consequently, the relationship between DNA replication and silencing remains an issue of debate. Here we analyze the effect of mutations in DNA replication factors (mcm5-461, mcm5-1, orc2-1, orc5-1, cdc45-1, cdc6-1, and cdc7-1) on the silencing of a group of reporter constructs, which contain different combinations of "natural" subtelomeric elements. We show that the mcm5-461, mcm5-1, and orc2-1 mutations affect silencing through subtelomeric ARS consensus sequences (ACS), while cdc6-1 affects silencing independently of ACS. orc5-1, cdc45-1, and cdc7-1 affect silencing through ACS, but also show ACS-independent effects. We also demonstrate that isolated nontelomeric ACS do not recapitulate the same effects when inserted in the telomere. We propose a model that defines the modes of action of MCM5 and CDC6 in silencing.     

A uniform procedure for the purification of CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1

We have established a uniform procedure for the expression and purification of the cyclin-dependent kinases CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. We attach a His₆-tag to one of the subunits of each complex and then co-express it together with the other subunits in Spodoptera frugiperda insect cells. The CDK complexes are subsequently purified by Ni²⁺-NTA and Mono S chromatography. This approach generates large amounts of active recombinant kinases that are devoid of contaminating kinase activities. Importantly, the properties of these recombinant kinases are similar to their natural counterparts (Pinhero et al. 2004, Eur J Biochem 271:1004–14). Our protocol provides a novel systematic approach for the purification of these three (and possibly other) recombinant CDKs. Published: August 18, 2004.     

Two methods for rapid assembly and oligomerization of synthetic genes: construction of human calcitonin-encoding sequences

Two fast, simple and reliable methods for construction of synthetic genes are described. The first represents a one-vial method for assembly and cloning of monomeric genes, and the second allows a simultaneous assembly, oligomerization and cloning of synthetic genes. Both methods consist of ligation of the synthetic oligodeoxyribonucleotides (oligos) building up the gene directly into the cloning vector, followed by screening of the recombinant clones for the presence of specific gene constructs. The average length of the oligomeric genes thus constructed is directly proportional to the molar ratio between the vector DNA and the synthetic oligos used. Both methods are illustrated by the construction of a series of human calcitonin-encoding genes.     

Glacial vicariance in Eurasia: mitochondrial DNA evidence from Scots pine for a complex heritage involving genetically distinct refugia at mid-northern latitudes and in Asia Minor

Background  

At the last glacial maximum, Fennoscandia was covered by an ice sheet while the tundra occupied most of the rest of northern Eurasia. More or less disjunct refugial populations of plants were dispersed in southern Europe, often trapped between mountain ranges and seas. Genetic and paleobotanical evidences indicate that these populations have contributed much to Holocene recolonization of more northern latitudes. Less supportive evidence has been found for the existence of glacial populations located closer to the ice margin. Scots pine (Pinus sylvestris L.) is a nordic conifer with a wide natural range covering much of Eurasia. Fractures in its extant genetic structure might be indicative of glacial vicariance and how different refugia contributed to the current distribution at the continental level. The population structure of Scots pine was investigated on much of its Eurasian natural range using maternally inherited mitochondrial DNA polymorphisms.     

Directional telomeric silencing and lack of canonical B1 elements in two silencer Autonomously Replicating Sequences in S. cerevisiae

Background

Detection of cell-free methylated DNA in plasma is a promising tool for tumour diagnosis and monitoring. Due to the very low amounts of cell-free DNA in plasma, analytical sensitivity is of utmost importance. The vast majority of currently available methods for analysing DNA methylation are based on bisulfite-mediated deamination of cytosine. Cytosine is rapidly converted to uracil during bisulfite treatment, whereas 5-methylcytosine is only slowly converted. Hence, bisulfite treatment converts an epigenetic modification into a difference in sequence, amenable to analysis either by sequencing or PCR based methods. However, the recovery of bisulfite-converted DNA is very poor.

Results

Here we introduce an alternative method for the crucial steps of bisulfite treatment with high recovery. The method is based on an accelerated deamination step and alkaline desulfonation in combination with magnetic silica purification of DNA, allowing preparation of deaminated DNA from patient samples in less than 2 hours.

Conclusions

The method presented here allows low levels of DNA to be easily and reliably analysed, a prerequisite for the clinical usefulness of cell-free methylated DNA detection in plasma.     

Sub-telomeric core X and Y' elements in S. cerevisiae suppress extreme variations in gene silencing

Telomere Position Effect (TPE) is governed by strong repression signals emitted by telomeres via the Sir2/3/4 Histone Deacetylase complex. These signals are then relayed by weak proto-silencers residing in the subtelomeric core X and Y' elements. Subtelomeres also contain Sub-Telomeric Anti-silencing Regions (STARs). In this study we have prepared telomeres built of different combinations of core X, Y' and STARs and have analyzed them in strains lacking Histone-Acetyltransferase genes as well as in cdc6-1 and Δrif1 strains. We show that core X and Y' dramatically reduce both positive and negative variations in TPE, that are caused by these mutations. We also show that the deletion of Histone-Acetyltransferase genes reduce the silencing activity of an ACS proto-silencer, but also reduce the anti-silencing activity of a STAR. We postulate that core X and Y' act as epigenetic "cushioning" cis-elements.     

Metal and Silicate Particles Including Nanoparticles Are Present in Electronic Cigarette Cartomizer Fluid and Aerosol

Background

Electronic cigarettes (EC) deliver aerosol by heating fluid containing nicotine. Cartomizer EC combine the fluid chamber and heating element in a single unit. Because EC do not burn tobacco, they may be safer than conventional cigarettes. Their use is rapidly increasing worldwide with little prior testing of their aerosol.

Objectives

We tested the hypothesis that EC aerosol contains metals derived from various components in EC.

Methods

Cartomizer contents and aerosols were analyzed using light and electron microscopy, cytotoxicity testing, x-ray microanalysis, particle counting, and inductively coupled plasma optical emission spectrometry.

Results

The filament, a nickel-chromium wire, was coupled to a thicker copper wire coated with silver. The silver coating was sometimes missing. Four tin solder joints attached the wires to each other and coupled the copper/silver wire to the air tube and mouthpiece. All cartomizers had evidence of use before packaging (burn spots on the fibers and electrophoretic movement of fluid in the fibers). Fibers in two cartomizers had green deposits that contained copper. Centrifugation of the fibers produced large pellets containing tin. Tin particles and tin whiskers were identified in cartridge fluid and outer fibers. Cartomizer fluid with tin particles was cytotoxic in assays using human pulmonary fibroblasts. The aerosol contained particles >1 µm comprised of tin, silver, iron, nickel, aluminum, and silicate and nanoparticles (<100 nm) of tin, chromium and nickel. The concentrations of nine of eleven elements in EC aerosol were higher than or equal to the corresponding concentrations in conventional cigarette smoke. Many of the elements identified in EC aerosol are known to cause respiratory distress and disease.

Conclusions

The presence of metal and silicate particles in cartomizer aerosol demonstrates the need for improved quality control in EC design and manufacture and studies on how EC aerosol impacts the health of users and bystanders.