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Identification of Small Molecular Weight Factors that Bind HeavyMetals in Algae

Objectives:  The objective of this proposal is to request funds to  modify our current high performance liquid chromatography (HPLC)  system so as to allow for the detection of cysteine-rich and  phytochelatin heavy metal binding complexes isolated from  Chlamydomonas reinhardtii. Phytochelatins (PC) are small molecular  weight, cysteine-rich peptides that bind heavy metals with high  affinity and specificity [Cai et al., 1999]. Recently, we have  engineered Chlamydomonas cells that have an increased capacity to  produce cysteine via introduction of the rice HAL2 gene into under  control of the beta-tubulin promoter. These algae bind three-fold more  cadmium than wild type when grown in the presence of cadmium  concentrations that induce PC synthesis. This is the first engineered  algae (to our knowledge) that has increased heavy metal binding  capacity when grown in the presence of heavy metal concentrations that  induce PC expression. As we have previously shown, heavy metal  concentrations that induce PC expression increase the metal binding  capacity of algae by more than 2,    fold [Cai et al., 1999]. It is  our hypothesis that algae expressing the HAL2 gene are capable of  synthesizing and accumulating more PC resulting in a greater capacity  to store toxic heavy metals. Alternatively, there may be other  cysteine-rich amino acids or proteins (e.g. glutathione, cysteine or  high molecular weight cysteine rich proteins) that bind the additional  cadmium in algae expressing the HAL2 gene. The most effective method  to isolate and characterize this diverse class of cysteine-rich, heavy  metal binding molecules is to separate them by HPLC and to  specifically detect the cysteine-rich molecules and/or phytochelatins  by derivitization with 5,5 -dithio-bis(2 nitrobenzoic acid) (DTNB)  [Grill et al., 1985].

Status
In progress
Type
Project
Start Date
End Date
Scope of Study
Laboratory Investigation
Scale of Phenomena
Physical/Chemical
Impact of Pollutants
Toxic Metals
State Province
Ohio
Annex Numbers
Hazardous Polluting Substances - lists
Specific Objectives for toxic substances and effluents
Surveillance and Monitoring

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