Technical advance: stringent control of transgene expression in Arabidopsis thaliana using the Top10 promoter system

We show that the tightly regulated tetracycline-sensitive Top10 promoter system (Weinmann et al. Plant J. 1994, 5, 559-569) is functional in Arabidopsis thaliana. A pure breeding A. thaliana line (JL-tTA/8) was generated which expressed a chimeric fusion of the tetracycline repressor and the activation domain of Herpes simplex virus (tTA), from a single transgenic locus. Plants from this line were crossed with transgenics carrying the ER-targeted green fluorescent protein coding sequence (mGFP5) under control of the Top10 promoter sequence. Progeny from this cross displayed ER-targeted GFP fluorescence throughout the plant, indicating that the tTA-Top10 promoter interaction was functional in A. thaliana. GFP expression was repressed by 100 ng ml-1 tetracycline, an order of magnitude lower than the concentration used previously to repress expression in Nicotiana tabacum. Moreover, the level of GFP expression was controlled by varying the concentration of tetracycline in the medium, allowing a titred regulation of transgenic activity that was previously unavailable in A. thaliana. The kinetics of GFP activity were determined following de-repression of the Top10:mGFP5 transgene, with a visible ER-targeted GFP signal appearing from 24 to 48 h after de-repression.     

Null alleles at the Huntington disease locus: implications for diagnostics and CAG repeat instability

PCR amplification of the CAG repeat in exon 1 of the IT15 gene is routinely undertaken to confirm a clinical diagnosis of Huntington disease (HD) and to provide predictive testing for at-risk relatives of affected individuals. Our studies have detected null alleles on the chromosome carrying the expanded repeat in three of 91 apparently unrelated HD families. Sequence analysis of these alleles has revealed the same mutation event, leading to the juxtaposition of uninterrupted CAG and CCG repeats. These data suggest that a mutation-prone region exists in the IT15 gene bounded by the CAG and CCG repeats and that caution should be exercised in designing primers that anneal to the region bounded by these repeats. Two of the HD families segregated null alleles with expanded uninterrupted CAG repeats at the lower end of the zone of reduced penetrance. The expanded repeats are meiotically unstable in these families, although this instability is within a small range of repeat lengths. The haplotypes of the disease-causing chromosomes in these two families differ, only one of which is similar to that reported previously as being specific for new HD mutations. Finally, no apparent mitotic instability of the uninterrupted CAG repeat was observed in the brain of one of the HD individuals.     

Optimal timing of cleft palate closure

Treatment objectives for the cleft palate patient--normal speech, normal maxillofacial growth, and normal hearing--are closely related. Controversy about the timing of cleft palate surgery is directed at the need for early palatoplasty for improved speech and hearing versus delayed hard palate repair for undisturbed facial growth. This controversy as to the value of early versus delayed closure continues into the present. The authors present an updated argument regarding this controversy along with a comprehensive literature review. They also present a logical algorithm based on the literature and their personal experience.     

The relationship of increased susceptibility of sperm DNA to denaturation and fertility in the stallion

The relationship between fertility and susceptibility of sperm DNA to denaturation was determined in a group of 84 actively breeding, clinically fertile stallions. Susceptibility of DNA to denaturation was determined using the sperm chromatin structure assay (SCSA). The SCSA measures, mean of alpha-t (mean alpha t), standard deviation of alpha-t (SD alpha t), and the COMP of alpha-t (cells outside the main population)] were significantly correlated with the percentage seasonal pregnancy rate (SPR; mean alpha t, r = -0.24, P < or = 0.05; % COMP alpha t, r = -0.27, P < or = 0.05); percentage pregnant per first cycle (FCP; SD alpha t, r = -0.30, P < or = 0.01; % COMP alpha t, r = -0.42, P < or = 0.0001); and the percentage pregnant per cycle (PC; mean alpha t, r = -0.31, P < or = 0.01; SD alpha t, r = -0.32, P < or = 0.01; % COMP alpha t, r = -0.41, P < or = 0.0001). This study describes detectable intrinsic variation in sperm chromatin structure among fertile stallions (SPR, mean = 83%; FCP, mean = 58%; PC, mean = 57%) in an active breeding population (number of mares bred/stallion/year, mean = 37), in the absence of overt reproductive abnormalities and apparent diseases such that an increase in the susceptibility of sperm DNA to denaturation is associated with reduced fertility, both in terms of efficiency of reproduction (FCP and PC) and seasonal pregnancy rate (SPR). Both COMP alpha t and mean alpha t were useful indicators of fertility, with COMP alpha t being the only SCSA value able to identify mean differences between fertility groupings for SPR and FCP, and overall it was the most reliable indicator of fertility in this group of stallions. The SCSA is able to evaluate a compartment of the spermatozoa which is different from that of traditional tests for sperm quality such as motility and morphology.     

Cytotoxicity-Associated Effects of Reactive Oxygen Species on Endothelin-1 Secretion by Pulmonary Endothelial Cells

In this study bovine pulmonary artery endothelial cells (BPAEC) were used as a model system to investigate the effects of the hypoxanthine–xanthine oxidase (HXXO) oxygen radical donor system on ET-1 secretion into pulmonary vasculature. Incubation of BPAEC with HXXO for 4 h caused a significant reduction in ET-1 secretion, which was significantly offset by allopurinol or catalase, but not by Cu/Zn superoxide dismutase (SOD). ET-1 secretion was also reduced by H₂O₂, and this effect was again significantly offset by catalase. XO alone also reduced ET-1 secretion, but to a significantly lesser degree than did HXXO, and this effect was not offset by allopurinol, catalase, or SOD. None of the oxidant treatments were associated with a loss of immunoreactive ET-1 from endothelial cell medium containing synthetic peptide. The HXXO- and H₂O₂-mediated reductions in ET-1 secretion were accompanied by evidence of reduced cell viability. This loss of viability was absent when cells were treated with HXXO + catalase, allopurinol, or mercaptopropionyl glycine, but not when SOD was present. We conclude that under conditions of oxidative stress, the pulmonary vascular endothelium responds by secreting less ET-1. This may be relevant to its vasodilator functions in the pulmonary vasculature, which would therefore be compromised when the endothelium is exposed to oxidant stress.     

Distribution of calmodulin protein and mRNA in growing pollen tubes

 Pollen tube growth is a vital process for angiosperm fertilisation and is dependent on the presence of a tip-focused gradient of cytosolic free calcium ([Ca²⁺]_c). In order to clarify some of the target molecules which convey the Ca²⁺ signal information, we investigated calmodulin distribution during tube growth. Fluorescently labelled calmodulin was pressure microinjected into pollen tubes and its distribution monitored by confocal microscopy. Calmodulin distributes evenly throughout the cell, but some of its binding sites form a V-shaped collar behind the apical region. This specific association dissipates upon growth arrest, and suggests an interaction of calmodulin with cytoskeletal-bound target proteins. The distribution of calmodulin mRNA was also analysed by microinjection of fluorescently labelled mRNA. No specific pattern was observed, with an even localisation in the body of tube and a lower concentration in the cell apex. Studies with localised application of inhibitors/activators indicate that calmodulin plays a crucial role in tip elongation but does not direct tube orientation. Received: 6 March 1998 / Revision accepted: 20 April 1998     

Traditionalism and Modernity in the Music and Dance of Oceania: Essays in Honour of Barbara B. Smith.

Traditionalism and Modernity in the Music and Dance of Oceania: Essays in Honour of Barbara B. Smith. Helen Reeves Lawrence and Don Niles. eds. Oceania Monograph 52. Sydney: University of Sydney, 2001. 267 pp.