Cloning, sequencing and characterization of a chitin synthase gene fragment from the fungus Phascolomyces articulosus /

Phascolomyces articulosus genomic DNA was isolated from 48 h old hyphae and was used for amplification of a chitin synthase fragment by the polymerase chain reaction method. The primers used in the amplification corresponded to two widely conserved amino acid regions found in chitin synthases of many fimgi. Amphfication resulted in four bands (820, 900, 1000 and 1500 bp, approximately) as visualized in a 1.2% agarose gel. The lowest band (820 bp) was selected as a candidate for chitin synthase because most amplified regions from other fimgi so far exhibited similar sizes (600-750 bp). The selected fragment was extracted from the gel and cloned in the Hinc n site of pUC19. The derived plasmid and insert were designated ^\5C\9'PaCHS and PaCHS respectively. The plasmid pUC19-PaC/fS was digested by several restriction enzymes and was found to contain BamHl and HincU sites. Sequencing of PaCHS revealed two intron sequences and a total open reading frame of 200 amino acids. The derived polypeptide was compared with other related sequences from the EMBL database (Heidelberg, Germany) and was matched to 36 other fiilly or partially sequenced fimgal chitin synthase genes. The closest resemblance was with two genes (74.5% and 73.1% identity) from Rhizopus oligosporus. Southern hybridization with the cloned fragment as a probe to the PCR reaction showed a strong signal at the fragment selected for cloning and weaker signals at the other two fragments. Southern hybridization with partially digested Phascolomyces articulosus genomic DNA showed a single band. The amino acid sequence was compared with sequences from other chitin synthase gene classes using the CLUSTALW program. The chitin synthase fragment from Phascolomyces articulosus was initially grouped in class n along with chitin synthase fragments from Rhizopus oligosporus and Phycomyces blakesleeanus which also belong to the same class, Zygomycetes. Bootstrap analysis using the neighbor-joining method available by CLUSTALW verified such classification. Comparison of PaCHS revealed conservation of intron positions that are characteristic of chitin synthase gene fragments of zygomycetous fungi.

Niagara River (Ontario) Remedial Action Plan fonds, 1984-1997, n.d.

The Niagara River Remedial Action Plan was part of an initiative to restore the integrity of the Great Lakes Basin ecosystem. In 1972, the Great Lakes Water Quality Agreement was signed by both Canada and the United States to demonstrate their commitment to protecting this valuable resource. An amendment in 1987 stipulated that Remedial Action Plans (RAPs) be implemented in 43 ecologically compromised areas known as Areas of Concern. The Niagara River was designated as one of these areas by federal and provincial governments and the International Joint Commission, an independent and binational organization that deals with issues concerning the use and quality of boundary waters between Canada and the United States. Although the affected area included parts of both the Canadian and American side of the river, Remedial Action Plans were developed separately in both Canada and the United States. The Niagara River (Ontario) RAP is a three-stage process requiring collaboration between numerous government agencies and the public. Environment Canada, the Ontario Ministry of the Environment, and the Niagara Peninsula Conservation Authority are the agencies guiding the development and implementation of the Niagara River (Ontario) RAP. The first stage is to determine the severity and causes of the environmental degradation that resulted in the location being designated an Area of Concern; the second stage is to identify and implement actions that will restore and protect the health of the ecosystem; and the third stage is to monitor the area to ensure that the ecosystem’s health has been restored. Stage one of the RAP commenced in January 1989 when a Public Advisory Committee (PAC) was established. This committee was comprised of concerned citizens and representatives from various community groups, associations, industries and municipalities. After several years of consultation, the Niagara River (Ontario) Remedial Action Plan Stage 2 Report was released in 1995. It contained 16 goals and 37 recommendations. Among them was the need for Canadians and Americans to work more collaboratively in order to successfully restore the water quality in the Niagara River. Stage three of the Niagara River (Ontario) RAP is currently ongoing, but it is estimated that it will be completed by 2015. At that point, the Niagara River Area of Concern will be delisted, although monitoring of the area will continue to ensure it remains healthy.