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All organisms in life need certain micro-elements to function, one of these elements is iron, and another copper. Whilst exploring the Iron transport system of S.cerevisiae Dancis et al (1994) happened upon a protein that somehow influences iron transport. This protein was then investigated and found to be a copper transporter, and was named copper transporter 1 (CTR1). Copper is an important cofactor in many processes, including the breakdown of superoxide and tyrosine, the oxidation of Cytochrome c and lysyl, and β-hydroxylation of dopamine (Linder 1991). Toxicity of copper though, is quite high in the presence of molecular oxygen, therefore copper concentration and transport is quite important to the organism (Halliwell and Gutteridge 1988). The genetic sequence was then characterized and it was clearly shown that a copper deficit results in an iron deficit. The identification and characterization of CTR1 revield the cDNA sequence, as well as chromosomal position and full sequence was obtained, as well as the roll of its protein, position of the protein, orientation in membrane and function was described.
Methods and Materials:
Screening of S.cerevisiae mutants incapable of Iron uptake (M3, M10 and strain 83)
Iron rich media
Plasmids pCM 106, pCM108
Yeast extract media (YE) containing non-fermentable carbon source
10 amino acid Myc epitope (Evan et al 1985)
Methods: gene identification
Selection scheme for yeast mutants where based on a HIS3 fusion construct, integration into the genome of S.cerevisciae with 609bp deleted from the HIS3 locus endogenous to the yeast. These where allowed to grow on yeast media, and the samples that showed iron-dependant histadine auxotrophy where selected for and incubated on a media rich in iron but in absence of histadine. This did not allow the cells to grow and after a week and a half papillae where found that represent mutants that repressed the iron-dependant auxotrophy for histadine. Two of these mutants M3 and M10 showed ferrous iron uptake deficiency. (Dancis et al 1994)
The M10 mutant was taken and crossed with another strain of genetic likeness (strain 83), generating diploids that where auxotrophic for His on Fe-enrich media plates, showing recessive phenotypic behaviour. M10 was used to isolate the wild-type allele that compliments the mutation of strain 83, and cloned back into a genomic library. Selection was done on YE and iron-rich plates to show ferric reduction. This allowed a colony to be identified that shows the expected phenotype, and plasmid rescue was done, shuttling the DNA into bacteria and then to yeast strain 83. This confirmed that the plasmid carried the gene to correct the abnormal phenotype seen in this strain, plasmid fragment was then purified and cut at unique SphI sites to linearize and used in URA3 targeting. The fragment was then genomically integrated int CM3260 (which has an ura3mutation), CM3260 was allowed to sporulate and the tetrads screened for uracil autotroph and non-repressed reductase on Fe-rich plates. (Dancis et al 1994)
Analysis of the sequence revieled only one open reading frame (ORF) needed for complementation. Frameshifts in the SalI site and SphI site (nt 187 and 440 respectively) showed loss of activity, the transcription site was mapped at -142, -172, -178 with regards to translation start site. Transcription termination was found to be at 230 nt into the 3' UTR by using Rapid amplification of cDNA ends (RACE) and primer extension techniques. To confirm identity a strain was created wih a large section of the underlying ORF (CTR1) deleted, and was found phenotypically similar to the M10 and M3 mutants. (Dancis et al 1994)
Methods: Protein analyses
SwissProt was used to search for the protein, predicting the amino acid sequence by using the ORF since the sequence itself did not show any real similarities to known sequence. This gave the result of a predicted protein with amino-groups and folds, it was shown to be similar to the CopA protein from Pseudomonas syringae and the Cop B from Entrobacter hirae, which are both copper-handelling proteins. This reconfirms the find that CTR1 a copper-handling protein. (Dancis et al 1994)
Localizing the protein was doen by epitope tagging, using the Myc epitope (Evan et al 1985), enserted into the ORF and shown to not deter protein functionality. A strain of yeast was transformed with a plasmid containing this tagged CTR1 and examined using imunoperoxidase under an electron microscope. (Dancis et al 1994)
Results showed that the protein CTR1 has copper-uptake and transport functions in the cell similar to that of Pseudomonas syringae CTR1, and that the copper uptake of the cells are inversely proportional to their ability to proliferate in high concentrations of copper and Iron in the media. It was also seen that copper uptake shows direct proportionality, as well as linkage to iron uptake, and that the one cannot function well without the other. Electron microscopy showed that the immune-stain pointed to the plasma membrane, highlighting that the CTR1 protein is membrane bound, moreover its carboxyl-end was in the cell itself. (Dancis et al 1994)
With the finding of the CTR1 locus in S.Cerevisciae a crucial step in the progress of science with regard to defining the relation between copper and iron uptake in bacteria as well as in plants and eukaryotes. It has also identified standards for copper binding motifs as well as conserved sequences that were used to find other genes of the similar function in various other organisms. Dancis et al (1994) showed the importance of a combination of techniques in molecular science, and how a different answer than expected is not necessarily the incorrect answer to a question.
CTR1, while being found to regulate copper uptake, was later found to not be the only Copper Transferase in the yeast cell. CTR 2 was soon discovered using sequence derived from a homologue in A.thaliana and hybridization assays (Kampfenkel et al 1995). More studies are done on copper transport, and soon enough the copper transport chain will be completely understood. This can provide us with novel insight into biological metabolism as a whole, be it prokaryotic or eukaryotic.