The effects of three chemical algaecides on cell numbers and toxin content of the cyanobacteria Microcystis aeruginosa and Anabaenopsis sp.

Toxic cyanobacteria blooms are a growing concern for public health and safety, due in part to the production of the hepatotoxin microcystin by certain species, including Microcystis aeruginosa. Management strategies for controlling cyanobacteria blooms include algaecide treatments, often with copper sulfate, and more recently oxidizers such as sodium percarbonate that produce hydrogen peroxide. This study assessed the effects of two copper-containing algaecides and one sodium percarbonate-containing algaecide on mitigating cell numbers and toxin content of cultured M. aeruginosa and summer (July) bloom samples of Anabaenopsis sp. in a brackish stormwater detention pond. Monitoring of the bloom revealed that Anabaenopsis sp. was associated with elevated levels of orthophosphate compared to nitrogen (dissolved inorganic nitrogen to phosphorus ratios were 0.19-1.80), and the bloom decline (September-October) was likely due to lower autumn water temperatures combined with potential grazing by the dinoflagellate Protoperidinium quinquecorne. Laboratory-based algaecide experiments included three dose levels, and cyanobacteria cell numbers and microcystin concentrations (particulate and dissolved) were evaluated over 7 d. Following exposure, copper-containing treatments generally had lower cell numbers than either sodium percarbonate-containing or control (no algaecide) treatments. Addition of algaecides did not reduce overall microcystin levels, and a release of toxin from the particulate to dissolved phase was observed in most treatments. These findings indicate that algaecide applications may visibly control cyanobacteria bloom densities, but not necessarily toxin concentrations, and have implications for public health and safety.

Multiple mechanisms influence regulation of the cystic fibrosis transmembrane conductance regulator gene promoter.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene is driven by a promoter that cannot alone account for the temporal and tissue-specific regulation of the gene. This has led to the search for additional regulatory elements that cooperate with the basal promoter to achieve coordinated expression. We previously identified two alternative upstream exons of the gene that were mutually exclusive of the first exon, and one of which showed temporal regulation in the human and sheep lung. We now demonstrate that this alternative splice product generates a stable protein, which initiates translation at an ATG in exon 4, and thus lacks the N terminus of CFTR. The other splice variant inhibits translation of the protein. In a search for the promoter used by the upstream exons, we identified a novel element that contributes to the activity of the basal CFTR promoter in airway epithelial cells, but does not function independently. Finally, we demonstrate that, in primary airway cells, skin fibroblasts, and both airway and intestinal cell lines, the CFTR promoter is unmethylated, irrespective of CFTR expression status. Thus, methylation is not the main cause of inactivation of CFTR transcription.

Stable integration of large PAC constructs in keratinocytes.

Transfer of P1-derived artificial chromosome (PAC) deoxyribonucleic acid (DNA) into keratinocytes is an extremely important technique that enables functional studies of keratinocyte-specific genes to be performed and genomic gene therapy for inherited and acquired diseases to be attempted. Ex vivo gene therapy approaches are possible using well-established conditions for keratinocyte culture and grafting, whilst the skin is the most accessible organ for administering in vivo therapy. PAC vectors lack relevant reporter genes to distinguish transfected mammalian cells from the non-transfected background, or to select clones in which the PAC construct has stably integrated into the genome. In this chapter, protocols to retrofit a reporter gene cassette will be described, together with techniques for transfecting large PAC constructs into keratinocytes without breakage. Protocols to select for stable integrants and to assess the integration event(s) within the keratinocyte genome will also be provided.

HNF1alpha is involved in tissue-specific regulation of CFTR gene expression.

The CFTR (cystic fibrosis transmembrane conductance regulator) gene shows a complex pattern of expression with tissue-specific and temporal regulation. However, the genetic elements and transcription factors that control CFTR expression are largely unidentified. The CFTR promoter does not confer tissue specificity on gene expression, suggesting that there are regulatory elements outside the upstream region. Analysis of potential regulatory elements defined as DNase 1-hypersensitive sites within introns of the gene revealed multiple predicted binding sites for the HNF1alpha (hepatocyte nuclear factor 1alpha) transcription factor. HNF1alpha, which is expressed in many of the same epithelial cell types as CFTR and shows similar differentiation-dependent changes in gene expression, bound to these sites in vitro. Overexpression of heterologous HNF1alpha augmented CFTR transcription in vivo. In contrast, antisense inhibition of HNF1 alpha transcription decreased the CFTR mRNA levels. Hnf1 alpha knockout mice showed lower levels of CFTR mRNA in their small intestine in comparison with wild-type mice. This is the first report of a transcription factor, which confers tissue specificity on the expression of this important disease-associated gene.

Quantitative methods for the analysis of CFTR transcripts/splicing variants.

In cystic fibrosis (CF), transcript analysis and quantification are important for diagnosis, prognosis and also as surrogate markers for some therapies including gene therapy. Classical RNA-based methods require significant expression levels in target samples for appropriate analysis, thus PCR-based methods are evolving towards reliable quantification. Various protocols for the quantitative analysis of CFTR transcripts (including those resulting from splicing variants) are described and discussed here.

A comparative genomic analysis of the cow, pig, and human CFTR genes identifies potential intronic regulatory elements.

The identification of sequences within noncoding regions of genes that are conserved between several species may indicate potential regulatory elements. This is important for genes with complex control mechanisms such as the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR demonstrates similar patterns of temporal and spatial expression in human and sheep, but these differ significantly in mouse cftr. The complete sheep CFTR sequence is unavailable so we annotated BAC clones encompassing the CFTR gene from two other artiodactyl species (cow and pig) for comparative sequence analysis. Regions of introns 2, 3, 10, 17a, 18, and 21 and 3' flanking sequence corresponding to human CFTR DNase I hypersensitive sites (DHS) showed high homology in the cow and pig. Cross-species sequence conservation also enabled finer mapping of other human DHS, including those in introns 1, 16, and 20. Additional potential regulatory elements not associated with human DHS were also identified.

Alternative splicing of the ovine CFTR gene.

Alternative splicing of the human CFTR gene was studied previously and shown not to generate functional CFTR-like chloride ion channels. However, it is possible that some of the alternatively spliced forms may encode CFTR proteins with different functions. The ovine CFTR gene is very similar to the human gene and has regulatory mechanisms in common. To evaluate whether the alternatively spliced forms of human CFTR are conserved in the sheep, the splice forms of the ovine CFTR gene were examined. A transcript lacking exon 9 was observed in the sheep, but unlike the human exon 9-transcript, it did not result from a polymorphic intron 8 splice acceptor site. Sheep CFTR transcripts lacking exon 17b were seen and have also been described in the human. Transcripts lacking 98 bp of the 5' end of exon 13, the whole of exon 13, and both exons 14b and 15 respectively were seen in sheep but have not been reported in human. Splice site donor and acceptor sequences were isolated, and alternative transcripts were shown to result from a combination of aberrant sites and competition of 5' splice donor sequences.

Evaluation of gene targeting by homologous recombination in ovine somatic cells.

Mouse models for some human genetic diseases are limited in their applications since they do not accurately reproduce the phenotype of the human disease. It has been suggested that larger animals, for example sheep, might produce more useful models, as some aspects of sheep physiology and anatomy are more similar to those of humans. The development of methods to clone animals from somatic cells provides a potential novel route to generate such large animal models following gene targeting. Here, we assess targeting of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in ovine somatic cells using homologous recombination (HR) of targeting constructs with extensive (>11 kb) homology. Electroporation of these constructs into ovine fetal and post-natal fibroblasts generated G418-resistant clones, but none analyzed had undergone HR, suggesting that at least for this locus, it is an extremely inefficient process. Karyotyping of targeted ovine fetal fibroblasts showed them to be less chromosomally stable than post-natal fibroblasts, and, moreover, extended culture periods caused them to senesce, adversely affecting their viability for use as nuclear transfer donor cells. These data stress the importance of donor cell choice in somatic cell cloning and suggest that culture time be kept to a minimum prior to nuclear transfer in order to maximize cell viability.