1. Open reading frame determination:
MacDNAsis utilized the cDNA of human cytochrome reductase to determine the possible open reading frames (orf). The largest open reading frame, highlighted in black (Fig. 1), was assumed to be the coding region of the human cytochrome reductase. The reading frame is contained within the amino acids numbered 544 and 2028.
Figure 1: MacDNAsis open reading frame analysis of human cytochrome reductase. The three different rows indicate the three different reading frame possibilities of the cDNA sequence. The start codons are represented by the red triangles, and the green lines represent the stop codons. The white areas between the triangles and the lines indicate open reading frames. The largest reading frame was assumed to be the coding region for the human cDNA which is highlighted black.
2. Molecular Weight Determination:
After MacDNAsis determined the orf, the molecular weight was calculated after translating the cDNA into the appropriate amino acids sequence of cytochrome reductase. The analysis predicted the molecular weight of cytochrome reductase to be 56672.8 daltons.
3. Determination of Cytochrome reductase hydropathy:
Using a Kyte and Doolittle plot, the hydrophobic and hydrophilic regions of the human cytochrome reductase protein were determined (fig 2).
Figure 2: Kyte and Doolittle analysis determined hydrophobic and hydrophilic regions of the coding sequence of human cytochrome reductase. Positive values on the x-axis, indicate hydrophobic regions while negative values indicate regions that are hydrophilic. The y-axis indicates the amino acids corresponding to the different hydrophobic and hydrophilic regions. Amino acid segments that are determined greater than 2, suggest possible integral membrane proteins. Cytochrome reductase has many hydrophobic regions, with the greatest hydrophilic region at 1-20 amino acids which could be a section of the protein that spans the inner mitochondria membrane(Fig. 2).
4. Hopp and Woods Antigenicity Plot of Cytochrome reductase:
Figure 3: Hopp and Woods antigenicity plot of cytochrome reductase
predicts areas that are hydrophilic. Hydrophilic regions which are
above the x-axis, are positive and therefore might be very antigenic.
Cytochrome is extremely variable, but the region between 325-360 amino
acids has high antigenicity potential and could be utilized to generate
a peptide to make a monoclonal antibody against (Fig 3).
5. Determination of Secondary Structure:
Next, the secondary structure of human cytochrome reductase
was predicted to help visualize the structure of the protein. The
secondary structure of a protein can be predicted by considering the likely
interactions of the amino acid side chains.
Figure 4: This analysis predicts the secondary structure of human cytochrome reductase. The blue bars represent alpha helixes, red striped bars represent beta strands, green bars represent turns in the structure, and black striped bars indicate coiled regions. This analysis indicates that human cytocrome reductase includes; about 16 alpha helixes, 23 beta strands, 7 turns in the structures, and 10 coiled regions. This prediction can be compared to a similar Rasmol image of a cytochrome reductase.
6. Multiple Sequence Alignment:
Using Higgins analysis, MacDNAsis compared five cytochrome reductase amino acid sequences from five species; including, Homosapien, Saccharomyces cerevisiae, Mus musculus, Drosophila melanogaster, and Arabindopsis thaliana. The sequences were provided by genebank, see my genebank search.
Figure 5: This analysis compares the amount of sequence similarity
that exists between five sequences of cytochrome reductase. The line labeled
human represents the amino acid sequence for Homo sapien. The line
labeled mouse represents the amino acid sequence for Mus musculus.
The line labeled plant represents the amino acid sequence for Arabindopsis
thaliana. The line labeled fly represents the amino acid sequence for
Drosophila melanogaster. The line labeled yeast represents the amino
acid sequence for Saccharomyces cerevisiae. Letters represent the
one letter abreviations for the amino acids. The highlighted amino acids
represent similar amino acids as compared to the other sequences listed.
The small horizontal lines in the sequences signify spaces in certain species
that helped maximize sequences alignment. The numbers on the left,
right, and above the sequences represent the corresponding amino acids.
As seen in the figure, similarity has been well conserved between the human
and mouse for the sequence corresponding to amino acids 40 - 50 (fig 5).
7. Phylogenetic Tree Analysis:
This phylogenetic tree of cytochrome reductase was assembled using the analysis of similarity determined from the sequence alignment above (fig 5).
Figure 6: Phylogenetic tree comparing the similarity in amino acid sequence of the five species; Homosapien, Saccharomyces cerevisiae, Mus musculus, Drosophila melanogaster, and Arabindopsis thaliana. The percentages indicate the amount of similarity between the sequences. The human and mice sequences have 34.8% similar amino acid sequences (Fig 6). While only a 4.9% similarity was determined between all five sequences. Evolutionary lineages can be predicted from these trees because people assume amino acid similarity can suggest conservation of amino acid sequence through evolution. Therefore, sequences that are less similar hypothetically diverged from the other sequences the earliest, or their mutation rate is quicker. Following this theory, this tree shows that yeast and fly share a more conserved cytochrome sequence than do flies and humans. However, little similarity was found between any of the structures suggesting that each sequence cytochrome reductase has changed significantly from the other species over time.