Introduction It is suggested through scientific research that all living organisms are connected- that we all share a common ancestor and that, through time, we have all evolved from the same starting point. Through evolution, different species have emerged, each different from the next. But although they are different, they still share much of the same genetic information. This evolution and connectedness is most clearly described as a tree- a Tree of Life (1) that branches off into many kingdoms and species, but all commencing from the same place. Phylogeny is the study of this tree and the interconnectedness of all species through the molecular sequencing of DNA and proteins. There are two main ways that genes are inherited among living things. (2) There is vertical gene transfer, directly from the parent to the offspring, and there is horizontal gene transfer, in which genes jump between two unrelated organisms. The latter can sometimes explain inconsistencies within phylogenic trees and why your hypothesis may not always match the results. Experiment / Hypothesis For this project, I chose to look at the Rh blood group gene. This gene can specify for the making of a number of different antigens, but mainly C, c, D, E and e. (3) For example, humans either have or do not have the RhD factor, which only refers to that specific antigen. Other species can have different
variations of this gene. However, the nucleotide sequences among these different species remains largely the same, which shows that the Rh genes have been greatly conserved through millions of years of evolution. The definite physiological function of these genes is not entirely clear, but it is thought that the RhD gene in humans has stayed so prominent because of its protection of infected humans against latent toxoplasmosis. It is also believed that the reason the RhD negative allele has stayed so prevalent in the population as well is that the protection against toxoplasmosis seems to be greater for RhD positive heterozygotes (3). I was interested in finding out the frequency of the Rh gene and its many variations, up to about 43 (3), across different species. I would expect that of course humans, and any of several species most closely related to humans such as monkeys and gorillas, would share this gene. I really wasn t sure what other species would also share it, although after discovering the gene s ability to protect against toxoplasmosis, I would not expect any animal in the cat family to carry it since cats are known to be the most prominent carriers of the infection. Material and Methods I began this inquiry with the NCBI website. From their homepage, I did a nucleotide search for the Rh gene. The first entry that came up was for the house mouse, accession number NM 011270.
After clicking on the entry, I formatted the information into FASTA. Then I did a BLAST by copying the nucleotide sequence, returning to the home page and selecting the BLAST heading, and specifying nucleotide BLAST under the Basic BLAST heading. Then, I pasted the FASTA sequence into the accession box and selected Others under the Database option. Once you click BLAST, the next page that appears will show you a list of species which share the gene and how similar their sequencing is to that of the species you started with. I then selected the ten different species with the highest similarity to the original species, and repeated the beginning steps of putting each one into FASTA format and copying and pasting their nucleotide sequences into a separate document. Once I got to this point, I was ready to begin using Clustal to align my sequences. After downloading the program, I made sure to delete any spaces and extra information from my document. All that remained were the common names of the species, without spaces, and the nucleotide sequences. In Clustal, I went into File, Load sequences, Alignment, Do complete alignment and then Align. At this time, all the sequences were aligned and upon scrolling right to left, I was able to find the point where all species began their alignment and where they ended aligning. I wrote down these numbers and went into Save sequences as, under File, and entered those numbers in the save from residue to. At this point, I was ready to exit Clustal.
Now I downloaded MEGA from the megasoftware.net website and saved it to the desktop. I then went under File and then Text editor and I opened my Clustal.aln document from the desktop. I had to delete all the extra stuff at the bottom of the page, such as astericks, etc. and then under Utilities, I converted it to MEGA format. I then saved this file to the desktop with an.meg extension on the file name. After then opening MEGA and the link to activate a data file, I opened my.meg file from the desktop and chose nucleotide sequence, yes to protein coding gene, and Standard genetic code. At this point, I was able to create different phylogeny trees in the new MEGA window that opened. I created a Minimum Evolution, a Maximum Parsimony and a Bootstrap test / Minimum Evolution tree. Results Each tree had the same groupings (in different orders), as follows: human / gibbon, orangutan / chimpanzee / gorilla, marmoset / capuchin, pig, and mouse / rat. The Bootstrap tree ranged from 98-100 on all nodes, which suggests high accuracy of the groupings. The trees are heavy on the monkey and ape families, so most of the species are relatively closely related. Trees: Minimum Evolution tree- Here I have shown the branching of the different species using a tree. Each branch represents each
branch of the phylogenic trees and splits accordingly. The leaves represent each different type of Rh gene present in that particular species. Maximum Parsimony- This tree explains the classifications of the species, along with their scientific names, common names, and images of each. Bootstrap Tree- This tree shows the accuracy numbers for each node. Discussion As I hypothesized, my results were heavy on the ape / monkey species, and of course, humans. It seemed a little surprising that mice and rats would carry it, although mouse DNA and humans is not far apart. It was surprising to me that the humans were grouped with the gibbon since chimpanzees, gorillas, humans and orangutans make up the great apes. After doing more research, I discovered a few other species that carry some version of this gene: zebrafish, marine sponge, African clawed frog, slime mold, earthworm, fruit fly, and green alga. Clearly this gene has stayed very prominent throughout evolution, even though there have been changes to it.
1. http://tolweb.org/tree/learn/concepts/whatisphylogeny.html 2. http://en.wikipedia.org/wiki/phylogenetics 3. http://en.wikipedia.org/wiki/rhesus_blood_group_system 4. http://