HAPPY mapping in a plant genome: reconstruction and analysis of a high-resolution physical map of a 1.9 Mbp region of Arabidopsis thaliana chromosome 4

HAPPY mapping is an in vitro approach for defining the order and spacing of DNA markers directly on native genomic DNA. This cloning-free technique is based on analysing the segregation of markers amplified from high molecular weight genomic DNA which has been broken randomly and 'segregated' by limiting dilution into subhaploid samples. It is a uniquely versatile tool, allowing for the construction of genome maps with flexible ranges and resolutions. Moreover, it is applicable to plant genomes, for which many of the techniques pioneered in animal genomes are inapplicable or inappropriate. We report here its demonstration in a plant genome by reconstructing the physical map of a 1.9 Mbp region around the FCA locus of Arabidopsis thaliana. The resulting map, spanning around 10% of chromosome 4, is in excellent agreement with the DNA sequence and has a mean marker spacing of 16 kbp. We argue that HAPPY maps of any required resolution can be made immediately and with relatively little effort for most plant species and, furthermore, that such maps can greatly aid the construction of regional or genome-wide physical maps.     

Drugs for the control of parasitic diseases: current status and development in schistosomiasis

The morbidity caused by schistosomiasis has been controlled in China, Egypt and the Philippines mainly by the widespread use of the safe and efficacious drug praziquantel (PZQ), and by oxamniquin and PZQ in Brazil. To date, there is no evidence of development of clinically relevant resistance. Thanks to the commitment of national governments at the World Health Assembly in 2001, and the emergence of significant funding for control, it is predicted that there will soon be more widespread use of PZQ in sub-Saharan Africa, where morbidity due to schistosomiasis is most prevalent. There are currently no available alternative drugs to PZQ (with the possible exception of oxamniquin), although perhaps PZQ analogues could be developed. Artemether, used to control malaria, is effective against immature schistosomes, but is less effective against adult worms. The efficacy of myrrh, recently marketed as an antischistosomacide in Egypt, has not been independently confirmed.     

Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

Background

Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive.

Results

We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy.

Conclusion

Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.     

Endcaps for stabilizing short DNA duplexes

The syntheses of endcaps for covalently linking the 3' and 5' hydroxyl groups of blunt end double-stranded DNA are described. Endcap diols were converted into DMTr protected phosphoramidites and incorporated between nucleotides 4 and 5 of a self-complementary octamer. The stabilizing effect of the endcaps on duplex DNA was determined by Tm experiments on the self-complementary octamer.     

The toxicity of textile reactive azo dyes after hydrolysis and decolourisation

The toxicity of C.I. Reactive Black 5 and three Procion dyes, as found in textile effluents, was determined using the bioluminescent bacterium Vibrio fischeri. Hydrolysed Reactive Black had a slightly greater toxicity than the parent form (EC(50) 11.4+/-3.68 and 27.5+/-4.01 mg l(-1), respectively). A baffled bioreactor with anaerobic and aerobic compartments was used to decolourise hydrolysed Reactive Black 5 in a synthetic effluent. Decolourisation of hydrolysed Reactive Black resulted in an increased toxicity (EC(50) 0.2+/-0.03 mg l(-1)). Toxicity was not detectable when decolourised Reactive Black 5 was metabolised under aerobic conditions. No genotoxicity was detected after the decolourisation of either the parent or the hydrolysed reactive dyes, either in vitro or in the bioreactor. The toxicity and genotoxicity of decolourised C.I. Acid Orange 7 was due to the production of 1-amino-2-naphthol (EC(50) 0.1+/-0.03 mg l(-1)).     

Splanchnic free fatty acid kinetics

These studies were conducted to assess the relationship between visceral adipose tissue free fatty acid (FFA) release and splanchnic FFA release. Steady-state splanchnic bed palmitate ([9,10-(3)H]palmitate) kinetics were determined from 14 sampling intervals from eight dogs with chronic indwelling arterial, portal vein, and hepatic vein catheters. We tested a model designed to predict the proportion of FFAs delivered to the liver from visceral fat by use of hepatic vein data. The model predicted that 15 +/- 2% of hepatic palmitate delivery originated from visceral lipolysis, which was greater (P = 0.004) than the 11 +/- 2% actually observed. There was a good relationship (r(2) = 0.63) between the predicted and observed hepatic palmitate delivery values, but the model overestimated visceral FFA release more at lower than at higher palmitate concentrations. The discrepancy could be due to differential uptake of FFAs arriving from the arterial vs. the portal vein or to release of FFAs in the hepatic circulatory bed. Splanchnic FFA release measured using hepatic vein samples was strongly related to visceral adipose tissue FFA release into the portal vein. This finding suggests that splanchnic FFA release is a good indicator of visceral adipose tissue lipolysis.     

J D Bernal (1901–1971) in perspective

Conclusion We must conclude that the sub-title of Bernal’s “The Social Function of Science” — “What science does: what science could do” is still the relevant challenge and indicates Bernal’s chief contribution, besides the foundation of molecular biology to our civilization. It is manifest that resources spent on armaments are a monstrous pathological symptom of our social structure. The ancient problem of “what is property” and what may be “owned” and by whom or by what organs of society is awakening.