Outgroup heteroduplex analysis using temperature gradient gel electrophoresis: high resolution, large scale, screening of DNA variation in the mitochondrial control region

The ability of DNA screening techniques such as Temperature Gradient Gel Electrophoresis (TGGE) and Heteroduplex Analysis to provide resolution approaching that provided by DNA sequencing for a fraction of the time, effort and expense point to them as the logical successor to allozyme electrophoresis for population genetics. Here we present a novel alternative to the standard TGGE/Heteroduplex Analysis protocol - Outgroup Heteroduplex Analysis (OHA). We assess this technique's sensitivity in comparison to previous screening approaches using a known hierarchy of sequence differences. Our data show that Outgroup Heteroduplex Analysis has greatly increased sensitivity for screening DNA variants from that of TGGE used alone and is easily applicable to large numbers of samples. Using this technique we can consistently detect differences of as small as one base change in a 433-base-pair fragment of Control Region mitochondrial DNA from Melomys cerbinipes (an Australian rodent). The approach should easily be extendable to nuclear loci and is not necessarily dependent on the use of a denaturing gradient When combined with a targeted sequencing effort, OHA provides a sensitive and simple means of obtaining allele/haplotype frequencies and their phylogenies for population and phylogeographic studies in molecular ecology.     

Distribution of damson-hop aphid (Phorodon humuli) migrants on hops in relation to hop variety and wind shelter

The distribution of migrant Phorodon humuli was studied in two hopgardens during June 1972. Migrants accumulating upon the young tissues near the bines' apices were counted. Plant size affected distribution, 100 % more migrants were found on 2-bine than on single bine strings and there was a positive linear relation between bine height and log₁₀ aphid numbers. Plants surrounding wire-trellis support poles were more heavily infested than plants not at poles, and migrants were more abundant on leeward orientated than windward strings. It is argued that the distribution of alatae reflected local patterns of wind shelter. Differences between plants declined in successive weeks. The influence of hop variety on migrant settling was studied in one of the hop-gardens. Fewer migrants settled on the variety Tolhurst than on Northern Brewer, and Fuggle was intermediate. Part, but not all, of the difference between varieties was explained by the larger average size of Northern Brewer plants. The observed aphid deposition rates in the two hop gardens were compared with hypothetical deposition rates calculated from numbers of P. humuli caught in a nearby 12-2 m Rothamsted Insect Survey suction-trap, and used to estimate flight durations. The estimates indicated that most infestation probably resulted from aphids within 1 h flying time of their primary host source.     

Heat shock temperature acclimation of normal secretory protein synthesis in barley aleurone cells

Exposure of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers to 40°C for a period of 3 h results in the selective suppression of the synthesis and secretion of hydrolytic enzymes; other normal cellular protein synthesis continues during heat shock. This suppression is correlated with secretory protein mRNA destabilization and the dissociation of stacked ER lamellae during heat shock (Belanger et al. 1986, Proceedings of the National Academy of Sciences USA 83, pp. 1354–1358). In this report we examined the effect of exposure to extended periods of heat shock. If exposure to 40°C was continued for a period of 18 h, the synthesis of α-amylase, the predominant secreted hydrolase, resumed. This was accompanied by increased α-amylase mRNA levels and the reformation of ER lamellae. Though initial exposure (3 h) to 40°C reduced protein secretion to ～10% of that observed in aleurone cells maintained at 25°C, exposure for prolonged periods (16–20 h) permitted the resumption of protein secretion to ～66% of non-heat-shocked control levels. The resumption of normal secretory protein synthesis during prolonged exposure to 40°C was correlated with an increase in the incorporation of [¹⁴C]glycerol into phosphatidylcholine and an increase in the ratio of saturated to unsaturated fatty acids in lipids isolated from ER membrane preparations. Increased fatty acid saturation has been demonstrated to enhance thermostability in biological membranes, and such changes in membrane composition may be important to the recovery of secretory protein synthesis at the ER.