The control of RNA synthesis in vitro

Conditions for the isolation and incubation of Xenopus cultured cell nuclei were optimised for maximal synthesis of RNA. The Xenopus nuclei showed all three RNA polymerase activities and appeared to process rRNA to 18S and 28S species, and possibly to initiate new RNA chains. The rate of RNA synthesis in isolated XTC-2 nuclei was changed when they were incubated with Xenopus oocyte and egg cell extracts in an optimised assay system (which included a preincubation step), the oocyte being stimulatory and the egg inhibitory. Cell extracts from other developmental stages assayed in the same way indicated that extracts of early cleavage stages were also inhibitory, whilst stages 7 ½ to 13 became increasingly stimulatory. Some possible trivial causes of these effects were eliminated. Four main stimulatory factors were isolated from mature oocytes and were purified to varying degrees by gel filtration and ion exchange chromatography. Three were found to be relatively non-specific in their action; the fourth specifically stimulated RNA polymerase I activity and the synthesis of rRNA. The activity of this factor was found to vary during oogenesis and early development (though possibly because of poor recovery), correlating reasonably with the known rates of rRNA synthesis in vivo. The factors were not spec les specific. A fifth minor stimulatory factor was found mainly to affect RNA polymerase II activity. The factor’s activity changed little between extracts from different developmental stages, however, this was the only purified factor which also stimulated RNA synthesis in isolated Xenopus blood nuclei. The factors stimulated elongation and possibly initiation also. All four triphosphates were required for factor stimulation to occur. Activity of the rRNA-specific factor was dependent on continued polymerase II activity. The factors stimulated RNA synthesis when assayed with Xenopus chromatin and added homologous polymerases. They were ineffective, however, when assayed with homologous purified DNA and RNA polymerases. Experiments involving the factors in vivo were inconclusive; their relevance and that of the nuclear assay system is discussed

Characterizing the Adaptive Optics Off-Axis Point-Spread Function - I: A Semi-Empirical Method for Use in Natural-Guide-Star Observations

Even though the technology of adaptive optics (AO) is rapidly maturing, calibration of the resulting images remains a major challenge. The AO point-spread function (PSF) changes quickly both in time and position on the sky. In a typical observation the star used for guiding will be separated from the scientific target by 10" to 30". This is sufficient separation to render images of the guide star by themselves nearly useless in characterizing the PSF at the off-axis target position. A semi-empirical technique is described that improves the determination of the AO off-axis PSF. The method uses calibration images of dense star fields to determine the change in PSF with field position. It then uses this information to correct contemporaneous images of the guide star to produce a PSF that is more accurate for both the target position and the time of a scientific observation. We report on tests of the method using natural-guide-star AO systems on the Canada-France-Hawaii Telescope and Lick Observatory Shane Telescope, augmented by simple atmospheric computer simulations. At 25" off-axis, predicting the PSF full width at half maximum using only information about the guide star results in an error of 60%. Using an image of a dense star field lowers this error to 33%, and our method, which also folds in information about the on-axis PSF, further decreases the error to 19%.Comment: 29 pages, 9 figures, accepted for publication in the PAS

Direct visualization of G-quadruplexes in DNA using atomic force microscopy

The formation of G-quadruplexes in G-rich regions of DNA is believed to affect DNA transcription and replication. However, it is currently unclear how this formation occurs in the presence of a complementary strand. We have used atomic force microscopy (AFM) to image stable RNA/DNA hybrid loops generated by transcription of the plasmid pPH600, which contains a 604-bp fragment of the murine immunoglobulin Sγ3 switch region. We show that the non-RNA-containing portion folds into G-quadruplexes, consistent with computational predictions. We also show that hybrid formation prevents further transcription from occurring, implying a regulatory role. After in vitro transcription, almost all (93%) of the plasmids had an asymmetric loop, a large asymmetric blob or a spur-like projection at the appropriate position on the DNA contour. The loops disappeared following treatment of the transcribed plasmid with RNase H, which removes mRNA hybridized with the template strand. Replacement of K+ in the transcription buffer with either Na+ or Li+ caused a reduction in the percentage of plasmids containing loops, blobs or spurs, consistent with the known effects of monovalent cations on G-quadruplex stability. The minimal sample preparation required for AFM imaging has permitted direct observation of the structural changes resulting from G-quadruplex formation

Over-use of thyroid testing in Canadian and UK primary care in frequent attenders : a cross-sectional study

Dr Greiver is supported through the Gordon F. Cheesbrough Research Chair in Family and Community Medicine from North York General Hospital.Background Thyroid stimulating hormone (TSH) is a common test used to detect and monitor clinically significant hypo- and hyperthyroidism. Population based screening of asymptomatic adults for thyroid disorders is not recommended. Objective The research objectives were to determine patterns of TSH testing in Canadian and English primary care practices, as well as patient and physician practice characteristics associated with testing TSH for primary care patients with no identifiable indication. Methods In this two-year cross-sectional observational study, Canadian and English electronic medical record databases were used to identify patients and physician practices. Cohorts of patients aged 18 years or older, without identifiable indications for TSH testing, were generated from these databases. Analyses were performed using a random-effects logistic regression to determine patient and physician practice characteristics associated with increased testing. We determined the proportion of TSH tests done concurrently with at least one common screening blood test (lipid profile or hemoglobin A1c). Standardized proportions of TSH test per family practice were used to examine the heterogeneity in the populations. Results At least one TSH test was done in 35.97 % (N=489,663) of Canadian patients and 29.36% (N=1,030,489) of English patients. Almost all TSH tests in Canada and England (95.69% and 99.23% respectively) were within the normal range (0.40-5.00 mU/L). A greater number of patient-physician encounters was the strongest predictor of TSH testing. 51.40% of TSH tests in Canada and 76.55% in England were done on the same day as at least one other screening blood test. There was no association between practice size and proportion of asymptomatic patients tested. Conclusions This comparative binational study found TSH patterns suggestive of over-testing and potentially thyroid disorder screening in both countries. There may be significant opportunities to improve appropriateness of TSH ordering in Canada and England and therefore improve allocation of limited system resources.PostprintPeer reviewe

Sequence analyses of three nuclear ribosomal loci and a mitochondrial locus in cytologically different forms of Thai Anopheles aconitus mosquitoes

Ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) of Anopheles aconitus mosquitoes were examined to investigate intra- and inter-species variation amongst the members of the Minimus group of Anopheles subgenus Cellia. Three rDNA loci (ITS1, ITS2 and D3 regions) and a mtDNA locus (cytochrome oxidase II) were analyzed in An. aconitus Form B and Form C collected in Chiang Mai Province, Thailand. The results show that the consensus sequences of the four loci of the two forms are consistent with those of mosquitoes in the genus Anopheles. No intraindividual variation was detected, but intrapopulation variation was present with polymorphic sequences in some forms for each gene examined. The variation rates were approximately 0.15 to 0.8%. These data indicate that An. aconitus Form B and Form C in Chiang Mai, Thailand are conspecific. In this study, the complete ITS1 sequence of An. aconitus is reported for the first time. The region showed a high variation rate (approximately 55%), compared to the closely related species An. minimus C. It is suggested that this rDNA locus may provide sequence information to differentiate the members of the Minimus group of Anopheles subgenus Cellia