Phylogenetic position of species in the genera Anoplophrya, Plagiotoma, and Nyctotheroides (Phylum Ciliophora), endosymbiotic ciliates of annelids and anurans

We have used small subunit rRNA gene sequences to determine the phylogenetic relationships of species in three genera of endosymbiotic ciliates. We have confirmed that the astome Anoplophrya marylandensis is related to ciliates in the Class Oligohymenophorea, supporting the view that astomes are derived from hymenostome-like ancestors. We confirmed that Plaglotoma lumbrici, formerly considered to be a heterotrich, is a stichotrich spirotrich ciliate most closely related to Paraurostyla weissei in this analysis. Thus, the somatic polykinetids of Plagiotoma can be concluded to be cirri. We report the details of our isolation of Nyctotheroides deslierresae and Nyctotheroides parvus and confirm previous reports that these clevelandellids are related to the metopid and caenomorphid ciliates, now placed in the Class Armophorea.     

High-resolution mapping of the Drosophila fourth chromosome using site-directed terminal deficiencies

For more than 80 years, the euchromatic right arm of the Drosophila fourth chromosome (101F-102F) has been one of the least genetically accessible regions of the fly genome despite the fact that many important genes reside there. To improve the mapping of genes on the fourth chromosome, we describe a strategy to generate targeted deficiencies and we describe 13 deficiencies that subdivide the 300 kb between the cytological coordinates 102A6 and 102C1 into five discrete regions plus a 200-kb region from 102C1 to 102D6. Together these deficiencies substantially improve the mapping capabilities for mutant loci on the fourth chromosome.     

Cometabolism of Trihalomethanes by Nitrosomonas europaea

The ammonia-oxidizing bacterium Nitrosomonas europaea (ATCC 19718) was shown to degrade low concentrations (50 to 800 μg/liter) of the four trihalomethanes (trichloromethane [TCM], or chloroform; bromodichloromethane [BDCM]; dibromochloromethane [DBCM]; and tribromomethane [TBM], or bromoform) commonly found in treated drinking water. Individual trihalomethane (THM) rate constants () increased with increasing THM bromine substitution, with TBM > DBCM > BDCM > TCM (0.23, 0.20, 0.15, and 0.10 liters/mg/day, respectively). Degradation kinetics were best described by a reductant model that accounted for two limiting reactants, THMs and ammonia-nitrogen (NH₃-N). A decrease in the temperature resulted in a decrease in both ammonia and THM degradation rates with ammonia rates affected to a greater extent than THM degradation rates. Similarly to the THM degradation rates, product toxicity, measured by transformation capacity (T_c), increased with increasing THM bromine substitution. Because both the rate constants and product toxicities increase with increasing THM bromine substitution, a water's THM speciation will be an important consideration for process implementation during drinking water treatment. Even though a given water sample may be kinetically favored based on THM speciation, the resulting THM product toxicity may not allow stable treatment process performance.     

Role of the cytoskeleton in sperm entry during fertilization in the freshwater bivalve Dreissena polymorpha

The present study examined the role of the cytoskeleton in sperm entry and migration through the egg cytoplasm during fertilization in the zebra mussel, Dreissena polymorpha (Bivalvia: Veneroida: Dreissenidae). Fertilization in this freshwater bivalve occurs outside the mantle cavity, permitting detailed observations of fertilization. After its initial binding to the egg surface, the sperm is incorporated in two stages: (1) a gradual incorporation of the sperm nucleus into the egg cortex, followed by (2) a more rapid incorporation of the sperm axoneme, and translocation of the sperm head through the egg cytoplasm. Initial incorporation into the egg cortex was shown to be microfilament dependent. Microfilaments were found in the sperm's preformed acrosomal filament, the microvilli on the egg surface, and in an actin-filled insemination cone surrounding the incorporating sperm. Treatment of eggs with cytochalasin B inhibited sperm entry in a dose- and time-dependent manner. Microtubule polymerization was not necessary for initial sperm entry. Following incorporation of the sperm head, the flagellar axoneme entered the egg cytoplasm and remained active for several minutes. Associated with the incorporated axoneme was a flow of cytoplasmic particles originating near the proximal end of the flagella. Inhibition of microtubule polymerization prevented entry of the sperm axoneme, and the subsequent cytoplasmic current was not observed. After sperm incorporation into the egg cortex, no appreciable microfilaments were associated with the sperm nucleus. A diminutive sperm aster was associated with the sperm nucleus during its decondensation, but no obvious extension toward the female pronucleus was observed. The sperm aster was significantly smaller than the spindle associated with the female pronucleus, suggesting a reduced role for the sperm aster in amphimixis.     

DNA repair of clustered uracils in HeLa cells

Two or more base damages, abasic sites or single-strand breaks (SSBs) within two helical turns of the DNA form a multiply damaged site (MDS) or clustered lesion. Studies in vitro and in bacteria indicate that attempts to repair two closely opposed base lesions can potentially form a lethal double-strand break (DSB). Ionizing radiation and chemotherapeutic agents introduce complex lesions, and the inability of a cell to repair MDSs is believed to contribute to the lethality of these treatments. The goal of this work was to extend the in vitro studies by examining MDS repair in mammalian cells under physiological conditions. Here, two opposing uracil residues separated by 3, 5, 7, 13 or 29 base-pairs were chosen as model DNA lesions. Double-stranded oligonucleotides containing no damage, a single uracil residue or the MDS were introduced into a non-replicating mammalian construct within the firefly luciferase open reading frame, or at the 5' or 3' end of the luciferase expression cassette. Following transient transfection into HeLa cells, luciferase activity was measured or plasmid DNA was re-isolated from the cells. Formation of a DSB was expected to decrease luciferase expression. However, certain single uracil residues as well as the MDSs decreased luciferase activity, which suggested that the reduction in activity was not due to DSB formation. In fact, Southern analysis of the re-isolated plasmid did not show the presence of linear DNA and demonstrated that none of the constructs was destroyed during repair. Further analysis of the re-isolated DNA demonstrated that only a small percentage of molecules originally carrying a single lesion or an MDS contained deletions. This work indicates that the majority of the clustered lesions were not converted to DSBs and that repair systems in mammalian cells may have established mechanisms to avoid the accumulation of SSB-repair intermediates.     

Scuticociliate endosymbionts of echinoids (phylum Echinodermata): phylogenetic relationships among species in the genera Entodiscus, Plagiopyliella, Thyrophylax, and Entorhipidium (phylum Ciliophora)

Morphological research on over 50 species of ciliates recorded as endosymbionts of echinoids suggests that invasion of the echinoid microhabitat occurred on at least 4 occasions. Gene sequence data confirm the phylogenetic distinctness of spirotrichean, armophorean, plagiopylean, and oligohymenophorean endosymbionts. It is also likely that oligohymenophoreans have repeatedly invaded the gut habitat. To test this hypothesis, we sequenced small subunit rRNA (SSrRNA) genes of 6 species representing the larger scuticociliate species found in the intestine of Strongylocentrotus pallidus from the northeast Pacific Ocean: Entodiscus borealis (Entodiscidae); Plagiopyliella pacifica and Thyrophylax vorax (Thyrophylacidae); and Entorhipidium pilatum, Entorhipidium tenue, and Entorhipidium sp. (Entorhipidiidae). SsrRNA genes were amplified by PCR, and sequences obtained in both directions. In all phylogenetic analyses, the scuticociliates are well supported as a clade. Entodiscus is distinct from these other echinoid taxa and is the sister taxon to the facultatively parasitic Uronema marinum. The other 5 echinoid species always form a clade whose basal species is the free-living Parauronema longum. The greatest genetic distance among these latter 5 species is less than 1.5%. This probably explains why the Thyrophylacidae and Entorhipidiidae are paraphyletic based on the SSrRNA gene sequences.     

An extremely rapid and simple DNA-release method for detection of M. tuberculosis from clinical specimens

A single-step, 5-min lysis method was investigated as a rapid technique to extract genomic DNA from mycobacteria for PCR detection of M. tuberculosis directly from clinical specimens. Of 67 smear-positive clinical specimens, 64 (95.5%) were positive by PCR after this rapid extraction method.