Molecular cloning and sequencing of ama-1, the gene encoding the largest subunit of Caenorhabditis elegans RNA polymerase II.

Two genomic sequences that share homology with Rp11215, the gene encoding the largest subunit of RNA polymerase II in Drosophila melanogaster, have been isolated from the nematode Caenorhabditis elegans. One of these sequences was physically mapped on chromosome IV within a region deleted by the deficiency mDf4, 25 kilobases (kb) from the left deficiency breakpoint. This position corresponds to ama-1 (resistance to alpha-amanitin), a gene shown previously to encode a subunit of RNA polymerase II. Northern (RNA) blotting and DNA sequencing revealed that ama-1 spans 10 kb, is punctuated by 11 introns, and encodes a 5.9-kb mRNA. A cDNA clone was isolated and partially sequenced to confirm the 3' end and several splice junctions. Analysis of the inferred 1,859-residue ama-1 product showed considerable identity with the largest subunit of RNAP II from other organisms, including the presence of a zinc finger motif near the amino terminus, and a carboxyl-terminal domain of 42 tandemly reiterated heptamers with the consensus Tyr Ser Pro Thr Ser Pro Ser. The latter domain was found to be encoded by four exons. In addition, the sequence oriented ama-1 transcription with respect to the genetic map. The second C. elegans sequence detected with the Drosophila probe, named rpc-1, was found to encode a 4.8-kb transcript and hybridized strongly to the gene encoding the largest subunit of RNA polymerase III from yeast, implicating rpc-1 as encoding the analogous peptide in the nematode. By contrast with ama-1, rpc-1 was not deleted by mDf4 or larger deficiencies examined, indicating that these genes are no closer than 150 kb. Genes flanking ama-1, including two collagen genes, also have been identified.

The soybean cyst nematode, Heterodera glycines: a genetic model system for the study of plant-parasitic nematodes.

Despite advances in understanding plant responses to nematode infection, little information exists regarding parasitic mechanisms. Recently, it has become possible to perform genetic analysis of soybean cyst nematode. Integration of classic and reverse genetics and genomic approaches for the parasite, with host genetics and genomics will expand our knowledge of nematode parasitism.

Experimental demonstration of the frequency shift of bandgaps in photonic crystal fibers due to refractive index scaling.

Experimental demonstration of the frequency shift of photonic bandgaps due to refractive index scaling using D2O-filled hollow-core photonic crystal fibers is presented. The results confirm a simple scaling law for bandgaps in fibers in which the low-index medium is varied.

Bend loss in all-solid bandgap fibres.

Experimental measurements of all-solid photonic bandgap fibres with an array of high-index rods in a low-index background revealed an unexpected variation of bend loss across different bandgaps. This behaviour was confirmed by calculations of photonic band structure, and explained with reference to the differing field distributions of the modes of the cladding rods. Our understanding was confirmed by further experiments, leading to proposals for the improvement of these fibres.

All solid photonic bandgap fiber based on an array of oriented rectangular high index rods.

We report the fabrication, characterization and modeling of an all-solid photonic bandgap fiber (PBGF) based on an array of oriented rectangular rods. Observed near-field patterns of cladding modes clearly identify the cut-off rod modes at the bandgap edges. The bend losses in this fiber depend on the bend direction, and can be understood by the directional coupling properties of the different rod modes and the modeled density of cladding states.

An improved photonic bandgap fiber based on an array of rings.

We describe the modeling, fabrication and characterization of a silica-core photonic bandgap fiber based on a 2-d array of raised-index cladding rings. The use of rings to form the cladding is shown to re-order the cladding modes in such a way as to broaden the photonic band gaps and reduce bend sensitivity. We compare the performance of the ring fiber with that of a similar fiber made using solid rods.

Design of low-loss and highly birefringent hollow-core photonic crystal fiber.

A practical hollow-core photonic crystal fiber design suitable for attaining low-loss propagation is analyzed. The geometry involves a number of localized elliptical features positioned on the glass ring that surrounds the air core and separates the core and cladding regions. The size of each feature is tuned so that the composite core-surround geometry is antiresonant within the cladding band gap, thus minimizing the guided mode field intensity both within the fiber material and at material/air interfaces. A birefringent design, which involves a 2-fold symmetric arrangement of the features on the core-surround ring, gives rise to wavelength ranges where the effective index difference between the polarization modes is larger than 10(-4). At such high birefringence levels, one of the polarization modes retains favorable field exclusion characteristics, thus enabling low-loss propagation of this polarization channel.

THE CAENORHABDITIS ELEGANS GENOME: A Guide in The Post Genomics Age.

The completion of the entire genome sequence of the free-living nematode, Caenorhabditis elegans is a tremendous milestone in modern biology. Not only will scientists be poring over data mined from this resource, but techniques and methodologies developed along the way have changed the way we can approach biological questions. The completion of the C. elegans genomic sequence will be of particular importance to scientists working on parasitic nematodes. In many cases, these nematode species present intractable challenges to those interested in their biology and genetics. The data already compared from parasites to the C. elegans database reveals a wealth of opportunities for parasite biologists. It is likely that many of the same genes will be present in parasites and that these genes will have similar functions. Additional information regarding differences between free-living and parasitic species will provide insight into the evolution and nature of parasitism. Finally, genetic and genomic approaches to the study of parasitic nematodes now have a clearly marked path to follow.

Chronic sulfonylurea therapy augments basal and meal-stimulated insulin secretion while attenuating insulin responses to sulfonylurea per se.

OBJECTIVE: To examine changes in glycemia and insulin secretion in response to SU per se and in response to a standard diet plus OD or TD SU therapy during chronic GP and GB therapy. RESEARCH DESIGN AND METHODS: Randomized (between agents and in order of dosing regimens), prospective, open, crossover study among 14 NIDDM patients to compare glucose, insulin, and C-peptide responses to a standard diet and to 10 mg of oral GP or GB taken without food 1) after 2 wk without therapy, 2) after 4 wk of either GP (n = 7) or GB (n = 7) treatment OD, and 3) after 4 wk of TD therapy with the same agent. Each patient received the same drug for maintenance therapy and for assessment of the response to the drug alone. RESULTS: We observed a comparable reduction in overall glycemia with both agents, with more marked postprandial effects for GP. Similar glucose, insulin, and C-peptide profiles for both agents during OD and TD therapy. Augmented insulin secretion in response to meals contrasting with reduced insulinotropic effects of the drugs per se with chronic therapy. CONCLUSIONS: Therapeutic equivalence of OD and TD dosing with GP and GB during chronic therapy. In view of the improved insulin secretion in response to nutrient stimuli, the attenuation of responses to SU per se during chronic therapy does not imply impairment of beta-cell secretory capacity or represent a therapeutic disadvantage.

DNA sequence and expression analysis of root-knot nematode-elicited giant cell transcripts.

Fifty-eight cDNA clones isolated from a library of transcripts exhibiting up regulation in tomato root giant cells induced by infection with the parasitic nematode Meloidogyne incognita were characterized. A survey of plant tissues identified 31 transcripts present in tissues other than root, including actively dividing and expanding tissues and mature leaf tissues. The identities of approximately 20% of the giant cell transcripts were inferred from DNA sequence data; they include sequences encoding a plasmalemma proton ATPase, a putative Myb-type transcription factor, and the largest subunit of RNA polymerase II.

Sequence comparison of the Caenorhabditis elegans dpy-13 and col-34 genes, and their deduced collagen products.

A 2232-nucleotide sequence spanning the col-34 gene from the nematode, Caenorhabditis elegans, is presented. This gene, which encodes a collagen protein (Clg), is transcribed from right to left with respect to the genetic map, and convergently with the nearby dpy-13 gene which also encodes a Clg. Both col-34 and dpy-13 have 5'-flanking elements in common with each other and also with other nematode Clg-encoding genes (clg). One element, variants of which are shared by col-7, col-19 and dpy-13, is predicted to be a target for a number of regulatory molecules, possibly including the ceh-18 product, a nematode POU-domain protein. The deduced amino acid sequence of Col-34 has a high degree of homology with the Dpy-13 collagen, although there are significant differences. In particular, one region of Dpy-13, which is predicted to have secondary structure different from Col-34, is altered by the recessive dpy-13(e225) mutation.

Effects of electromagnetic fields on photophasic circulating melatonin levels in American kestrels.

Birds reproduce within electromagnetic fields (EMFs) from transmission lines. Melatonin influences physiologic and behavioral processes that are critical to survival, and melatonin has been equivocally suppressed by EMFs in mammalian species. We examined whether EMFs affect photophasic plasma melatonin in reproducing adult and fledgling American kestrels (Falco sparverius), and whether melatonin was correlated with body mass to explain previously reported results. Captive kestrel pairs were bred under control or EMF conditions for one (short-term) or two (long-term) breeding seasons. EMF exposure had an overall effect on plasma melatonin in male kestrels, with plasma levels suppressed at 42 days and elevated at 70 days of EMF exposure. The similarity in melatonin levels between EMF males at 42 days and controls at 70 days suggests a seasonal phase-shift of the melatonin profile caused by EMF exposure. Melatonin was also suppressed in long-term fledglings, but not in short-term fledglings or adult females. Melatonin levels in adult males were higher than in adult females, possibly explaining the sexually dimorphic response to EMFs. Melatonin and body mass were not associated in American kestrels. It is likely that the results are relevant to wild raptors nesting within EMFs.

Simultaneous quantification of progesterone, estrone, estradiol-17 beta and corticosterone in female American kestrel plasma.

Progesterone, estrone, estradiol-17 beta and corticosterone were quantified simultaneously for the first time in female American kestrel (Falco sparverius) plasma. A mean level for each hormone was determined for the laying and non-laying periods of the summer (April-September), and for February. Means were comparable to those of other wild avian species and were significantly higher for the laying period than for the other 2 periods. The mean corticosterone level for February was higher than that for the non-laying summer period. Plasma from laying kestrels, unlike that from other avian species, required lipid removal before column chromatography. Of 2 lipid removal techniques compared, i.e. the cold methanol and hexane:methanol techniques, the latter proved superior.

Behavior of free-ranging and captive American kestrels under electromagnetic fields.

Wild birds, particularly raptors, commonly use electrical transmission structures for nesting, perching, hunting, and roosting. Consequently, birds are exposed to electromagnetic fields (EMFs). The amount of time that wild reproducing American kestrels (Falco sparverius) were exposed to EMFs was determined, and the effects of EMFs on the behavior of captive reproducing kestrels were examined. Wild kestrels were exposed to EMFs for 25% to 75% of the observed time. On a 24-h basis, estimated EMF exposure of wild kestrels ranged from 71% during courtship, to 90% during incubation, similar to that experienced by captive kestrels (88% of a 24-h period). Additionally, captive kestrels were exposed to EMF levels experienced by wild kestrels nesting under 735-kV power lines. Captive EMF females were more active, more alert, and perched on the pen roof more frequently than control females during courtship. EMF females preened and rested less often during brood rearing. EMF male kestrels were more active than control males during courtship, and more alert during incubation. Increased activity of kestrels during courtship may be linked to changes in corticosterone, but likely not melatonin. Observed behavioral changes were unlikely to directly result in the better growth of nestlings and fledging success, or poorer hatching success, of the EMF group, as previously reported. Behavioral changes of captive EMF kestrels may be observed in wild kestrels.

Changes in egg composition of American kestrels exposed to dietary polychlorinated biphenyls.

Changes in the quality of eggs of birds exposed to polychlorinated biphenyls (PCBs) have been described, but have never been directly attributed to PCBs. Polychlorinated biphenyl residues in eggs have been associated with reduced reproductive success and embryonic deformities in wild birds. Egg size and composition, specifically the amount of albumen, yolk, and water in an egg, also influence the growth and viability of embryos and hatchlings, and consequently the reproductive success of birds. To deter mine whether PCB exposure of adult birds affected the size and composition of their eggs, 25 pairs of captive American kestrels (Falco sparverius) were fed a mixture of PCB-spiked (1248:1254:1260) food to give an approximate exposure of 7 mg/kg body weight/d, beginning 1 mo prior to pairing, and continuing throughout the courtship, egg-laying, and incubation periods. This dietary level in the adult female kestrels resulted in mean total PCB residues in the eggs of 34.1 microg/g wet weight (geometric mean), which is environmentally relevant. PCB residues in eggs increased with the time of female exposure to the contaminated diet and laying date. Variation in egg size within PCB clutches was significantly greater than within control clutches, although absolute egg mass and volume did not differ markedly by treatment. Only infertile eggs and only one egg per clutch were used for egg composition analysis. Yolks in the PCB-contaminated eggs were heavier, with less wet and dry albumen relative to control eggs. Water content and eggshell thickness were not significantly affected by PCB exposure. These results suggest that eggs from the PCB treatment have relatively more lipid and less protein available for embryonic development. Changes in egg composition were not associated with egg size, lay date, ambient temperature, humidity, or precipitation, which are factors known to affect these variables in bird eggs. The PCB-induced changes in egg composition described here provide insight into possible mechanisms contributing to reduced reproductive performance in wild birds exposed to PCBs.

Influence of a diet of fluoride-fed cockerels on reproductive performance of captive American kestrels.

In 1982, twenty-four pairs of captive American kestrels (Falco sparverius) were forced to renest by removal of their first clutches 6 days after their completion. Immediately following, each of three groups of eight pairs was randomly assigned to one of three daily dietary regimes for 10 days: (1) three 1-day old cockerels with background levels of F(-) (62.4+/-51ppm, mean+/-SD) in their femurae, (2) two 10-day old cockerels with 4512+/-810ppm of F(-) in their femurae, (3) two 10-day old cockerels with 7690+/-417ppm of F(-) in their femurae. Fluoride levels in femurae of treated kestrels were significantly (P<0.0025) higher than those of control birds. Clutch sizes tended to be smaller as more fluoride was added to the diet, but not significantly so, due to an increase of the variance in the treatment group. Per cent fertility and per cent hatchability were not significantly affected by treatment. The fluoride content in eggshells in the fluoride-treated groups differed significantly from those of the control group (P<0.001).

Effects of electromagnetic fields on the reproductive success of American kestrels.

Reduced reproductive success of birds nesting near power lines has been documented but never directly attributed to electromagnetic fields (EMFs). Laboratory studies have identified EMF effects on embryonic development, but reproductive success of wild birds is dependent on additional factors, including fertility, egg size, hatching, and fledging success. We tested whether EMFs affect reproductive success of birds. Captive American kestrels (Falco sparverius) were bred for one season per year for 2 yr under either controlled or EMF conditions. EMF exposure was equivalent to that experienced by wild reproducing kestrels and was weakly associated with reduced egg laying in 1 yr only. In both years fertility was higher, but hatching success was lower in EMF pairs than control pairs. Fledging success was higher in EMF pairs than control pairs in 1995 only. Egg composition and embryonic development were examined in 1 yr only, but hatchlings were measured in both years. EMF eggs were larger, with more yolk, albumen, and water, but had thinner egg shells than control eggs. Late-term EMF embryos were larger and longer than control embryos, although hatchlings were similar in body mass and size. EMF exposure affected reproductive success of kestrels, increasing fertility, egg size, embryonic development, and fledging success but reducing hatching success.

Manipulation of host gene expression by root-knot nematodes.

Root-knot nematodes (Meloidogyne spp.) establish elaborate feeding sites in their host. Unique patterns of gene expression are induced in root cells, resulting in formation of a novel cell type called a giant cell. Based on analysis of approximately 220 giant cell expressed genes, key elements of giant cell function and regulation have been identified; examples are discussed in the context of giant cell biology and ontogeny. The potential to effect nematode control by manipulating these genes in transgenic host plants is considered, and models for giant cell induction are presented.

Rapid miniprep isolation of mitochondrial DNA from metacestodes, and free-living and parasitic nematodes.

A method, based on one to isolate supercoiled plasmid DNA from bacterial cells, has been developed to purify mitochondrial DNA (mtDNA) from cestode and nematode tissue easily and efficiently. Starting with as little as 100 mg of helminth tissue, sufficient mtDNA for electrophoretic analysis was extracted. This DNA was essentially free of nuclear DNA and readily digested by restriction endonucleases. Approximately 20% of the mtDNA in helminth tissue was recovered, which is a significant improvement over previously available techniques.

Reproduction success of American kestrels exposed to dietary polychlorinated biphenyls.

While reproduction of wild birds is adversely affected by multiple environmental contaminants, we determined that polychlorinated biphenyls (PCBs) alone alter reproduction. Captive American kestrels (Falco sparverius), fed PCB-spiked (Aroclor 1248:1254:1260) food (7 mg/kg body weight/d) prior to and during the first breeding season only (100 d) laid eggs with environmentally relevant levels of total PCBs (34.0 microg/g whole egg wet wt vs 0 microg/g for controls). Reproduction changed during, not after, PCB exposure in this two-year study. The PCB-exposed pairs laid smaller clutches later in the season and laid more totally infertile clutches. Hatching success was reduced in PCB-exposed pairs, and 50% of PCB nestlings died within 3 d of hatching. Nearly 60% of PCB-exposed pairs with hatchlings failed to produce fledglings. Higher levels of total PCB residues and congeners were associated with later clutch initiation and fewer fertile eggs, hatchlings, and fledglings. We suggest that nonpersistent PCB congeners have a greater influence on reproduction than do persistent congeners.