Rapid high-efficiency site-directed mutagenesis by the phosphorothioate approach.

Several improvements to the existing phosphorothioate-based site-directed mutagenesis methodology are reported, and here it is demonstrated that the new procedure is able to produce large deletions, insertions and point mutations rapidly and with very high efficiency. The time required for the polymerization step has been reduced by using T7 DNA polymerase to extend the mutant oligonucleotide primer-template. The reaction produces good yields of double-stranded closed-circular DNA and some partially polymerized template. The reaction was treated with T5 D15 exonuclease to selectively destroy partially polymerized single-stranded phage DNA that may otherwise contribute to an increased background of wild-type transformants. The use of these enzymes greatly facilitates the implementation of the phosphorothioate-based site-directed mutagenesis method by requiring less template DNA and by allowing all the in vitro manipulations to be completed in a day. In its present form the method may easily be automated, enabling large systematic site-directed mutagenesis projects to be undertaken.     

Strategies to Guide the Return of Genomic Research Findings: An Australian Perspective

In Australia, along with many other countries, limited guidance or other support strategies are currently available to researchers, institutional research ethics committees, and others responsible for making decisions about whether to return genomic findings with potential value to participants or their blood relatives. This lack of guidance results in onerous decision-making burdens—traversing technical, interpretative, and ethical dimensions—as well as uncertainty and inconsistencies for research participants. This article draws on a recent targeted consultation conducted by the Australian National Health and Medical Research Council to put forward strategies for supporting return of finding decision-making. In particular, we propose a pyramid of decision-making support: decision-making guidelines, technical and interpretative assistance, and ethical assistance for intractable “tough” cases. Each step of the pyramid involves an increasing level of regulatory involvement and applies to a smaller subsection of genomic research findings. Implementation of such strategies would facilitate a growing evidence base for return of finding decisions, thereby easing the financial, time, and moral burdens currently placed on researchers and other relevant decision-makers while also improving the quality of such decisions and, consequently, participant outcomes.     

The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA

The RF IV form of M13 DNA was synthesized enzymatically in vitro, using the viral (+)strand as template, to contain phosphorothioate-modified internucleotidic linkages of the Rp configuration on the 5' side of every base of a particular type in the newly-synthesized (-)strand. Twenty nine restriction enzymes were then tested for their reactions with the appropriate modified DNA types having a phosphorothioate linkage placed exactly at the cleavage site(s) of these enzymes in the (-)strand. Eleven of the seventeen restriction enzymes tested that had recognition sequences of five bases or more could be used to convert the phosphorothioate DNA entirely into the nicked form, either by simply allowing the reaction to go to completion with excess enzyme (Ava I, Ava II, Ban II, Hind II, Nci I, Pst I or Pvu I) or by stopping the reaction at the appropriate time before the nicked DNA is linearized (Bam HI, Bgl I, Eco RI or Hind III). Only modification of the exact cleavage site in the (-)strand could block linearization by the first class of enzymes. The results presented imply that the restriction enzyme-directed nicking of phosphorothioate M13 DNA occurs exclusively in the (+)strand.     

Molecular marker development and linkage analysis in three low phytic acid barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis>) mutant lines

Phytate is the primary form of phosphorus found in mature cereal grain. This form of phosphorus is not available to monogastric animals due to a lack of the enzyme phytase in their digestive tract. Several barley low phytic acid (lpa) mutants have been identified that contain substantial decreases in seed phytate accompanied by concomitant increases in inorganic phosphorus. Seed homozygous for low phytic acid 1-1 (lpa1-1) or low phytic acid 2-1 (lpa2-1) has a 50% and 70% decrease in seed phytate respectively. These mutations were previously mapped to chromosomes 2HL and 7HL respectively. The RFLP marker ABC153 located in the same region of 2H was converted to a sequence-characterized-amplified-region (SCAR) marker. Segregation analysis of the CDC McGwire × Lp422 doubled haploid population confirmed linkage between the SCAR marker and the lpa1-1 locus with 15% recombination. A third low phytic acid mutant, M635, has a 75% decrease in phytate. This mutation was located to chromosome 1HL by linkage with an inter-simple sequence repeat (ISSR) based marker (LP75) identified through bulked-segregant analysis, and has been designated lpa3-1. Based on analysis of recombination between marker LP75 and low phytic acid in an additional mutant line M955 (95% phytate decrease), lpa3-1 and the mutation in M955 are in the same region on chromosome 1HL, and may be allelic.     

Range expansion of a selfing polyploid plant despite widespread genetic uniformity

Background and Aims

Ongoing and previous range expansions have a strong influence on population genetic structure of plants. In turn, genetic variation in the new range may affect the population dynamics and the expansion process. The annual Ceratocapnos claviculata (Papaveraceae) has expanded its Atlantic European range in recent decades towards the north and east. Patterns of genetic diversity were investigated across the native range to assess current population structure and phylogeographical patterns. A test was then made as to whether genetic diversity is reduced in the neophytic range and an attempt was made to identify source regions of the expansion.

Methods

Samples were taken from 55 populations in the native and 34 populations in the neophytic range (Sweden, north-east Germany). Using amplified fragment length polymorphism markers an analysis was made of genetic variation and population structure (Bayesian statistical modelling) and population differentiation was quantified. Pollen/ovule ratio was analysed as a proxy for the breeding system.

Key Results

Genetic diversity at population level was very low (mean H_e = 0·004) and two multilocus genotypes dominated large parts of the new range. Population differentiation was strong (F_ST = 0·812). These results and a low pollen/ovule ratio are consistent with an autogamous breeding system. Genetic variation decreased from the native to the neophytic range. Within the native range, H_e decreased towards the north-east, whereas population size increased. According to the Bayesian cluster analysis, the putative source regions of the neophytic range are situated in north-west Germany and adjacent regions.

Conclusions

Ceratocapnos claviculata shows a cline of genetic variation due to postglacial recolonization from putative Pleistocene refugia in south-west Europe. Nevertheless, the species has expanded successfully during the past 40 years to southern Sweden and north-east Germany where it occurs as an opportunistic neophyte. Recent expansion was mainly human-mediated by single long-distance diaspore transport and was facilitated by habitat modification.     

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

The organization and control of grooming in cats

Grooming in small felids has received little attention compared with grooming in rodents, bovids and primates where grooming is also common. This study set out to describe the general pattern, time budget and degree of cephalocaudal sequencing of self-oral grooming in the domestic cat. In 11 cats confined for the purposes of videotaping, sleeping and resting accounted for 50% of the time budget. Oral grooming, 91% of which was to multiple body regions, accounted for 4% of the overall time budget or 8% of non-sleeping/resting time. Scratch grooming, always directed to single regions, occupied about 1/50 of the time of oral grooming. There was a moderate and significant cephalocaudal trend to grooming. An increased likelihood for oral grooming to follow periods of sleep or rest was indicated by a significant negative correlation between sleep/rest duration and latency to the subsequent grooming bout. The effect of enforced deprivation of grooming on the subsequent occurrence of grooming was explored by the 3-day application of Elizabethian collars, which prevented oral grooming or control collars that did not prevent grooming. In the 12 h immediately after removal of the Elizabethian collars, oral grooming increased by 67% and scratch grooming by 200% compared with the grooming rate after removal of control collars. By the second 12 h, the apparent catch-up effect of grooming had disappeared. The occurrence of cephalocaudally-directed, multiple-region oral grooming and deprivation-enhanced grooming would appear to represent aspects of a central control mechanism for the organization and regulation of grooming.