The Attractin Gene

I was excited to finally obtain data on Wednesday after the days of PCR, gel electrophoresis, PCR clean-up, and nanodropping, and to learn how to analyze that data. The data is received electronically, and can be analyzed on a program called Sequencher. This program allows you to align the forward and reverse sequences for the DNA from each specimen and locate regions of variability between the two sequences. The sequencing center sequences each gene as if it were as long as the longest gene submitted on the plate of DNA samples; thus, the end of each fragment contains numerous “N”s, meaning there was no nucleotide base in those regions and the end of the sequence can be removed. Looking at the remaining sequence in the form of a chromatogram, a graph that represents the presence of each nucleotide base at each region of the gene by showing each nucleotide base as a distinctly colored peak, the more reliable data can be preserved, while the less reliable data can be eliminated. When the cited nucleotide bases are reliable, the peaks on the graph are distinct; when the cited nucleotide bases are unreliable, the peaks on the graph are less distinct and are overlapping. The cited nucleotide bases are typically less accurate at the beginning and end of the sequence, and those regions are thus eliminated. The next step is to examine the pairs of sequences to detect regions of variability and determine whether there is actually a likely variation in base pairs, and if so, whether the variation is due to the presence of two varying alleles (versions of the gene), creating heterozygotes (individuals with two versions of a gene). Finally, the combined forward and reverse sequences for each specimen is placed on the same page and variation among the specimen can be detected. This variation is then analyzed to determine its association with coat color.

While only about fifteen of the thirty-one samples from the Kenzin population of mice were sent for sequencing, the findings are already promising! The sequenced DNA revealed that there are heterozygotes in this population, specifically in two distinct regions of intron 17; these regions contained SNPs (single nucleotide polymorphisms, or nucleotide base differences within a population). The variability in these regions is in perfect linkage disequilibrium, meaning there is a non-random association of alleles (versions of the same gene) at the two loci (regions of the gene). There is also another site of variability among the samples in the sequenced intron that is likely unrelated to the previous two sites. However, while the samples sequenced included only one light agouti animal, no melanic (dark) animals, and a majority of dark agouti animals, the variation appeared among the dark agouti animals, revealing the likelihood that there is no association between variation in this region of attractin and coat color in these mice. (The pigmentation caused by the agouti gene is the banded pattern of light and dark color on the hair.) This is a positive finding, as it further solidifies the likelihood that the agouti gene, which is located downstream of the attractin gene on the same chromosome as attractin, is responsible for coloration in these mice and could be a site on which natural selection is acting (creating coat color beneficial for camouflage and thus survival); this assumption is because association between genetic variation and coat color decays at attractin (upstream of agouti), and thus the variation responsible for coat color must be located downstream of intron 17 of attractin. Hopefully, the remaining sequences from this population, which should be sent to the sequencing center either today or next Monday, will correlate with the above data.

In addition to receiving and analyzing this data, I continued active experimentation within the laboratory, continued reading various articles, and discussed the future goals of my project and the overall focus of the laboratory with Professor Nachman and the other individuals studying coat color variation. I completed the previously described steps necessary for sequencing intron 17 in approximately ten individuals from the Carrizozo site before the meeting to discuss immediate versus long-term goals of the laboratory. A major goal of the laboratory will be to organize the specimen based on coat color to confirm that they are categorized correctly. This will begin by three of the four individuals, including myself, who are working on the coat color project placing the Kenzin specimen into bins depicting three classes of color (light, dark, and intermediate) both in the light and dark (the animals’ natural environment) independently of one another in order to determine if our visual categorizations of phenotype (appearance) correlate. The animals must also be classified by light transmittance through hair using a spectrophotometer, which is currently in the mail to be updated and repaired. Finally, the length of the bands on the hair of each specimen must be measured by two individuals (to create greater accuracy). These many processes will ensure that when there is genetic variation among the samples, we can accurately determine the association of that variation with coat color. However, current sequencing within the agouti gene has revealed genetic variation among light and dark agouti animals that is not associated to variation in those two coat colors (although genetic variation is highly associated to the variation between agouti and melanic pigmentation), proposing the question of whether the intermediate color of the specimen actually serves a functional purpose for survival. Another main goal of the laboratory is to fully sequence the agouti gene in one specimen of each of the three phenotypes of the Kenzin population to locate any regions of variation that could then be more greatly examined among all of the Kenzin samples. For my project involving the attractin gene, due to the findings in the Kenzin population, I will begin to more greatly examine the role of the attractin gene in coloration in the Carrizozo population. Additionally, because the attractin gene is so large, I will design primers to sequence the gene at approximately twenty kilobase-pair intervals in order to find regions of variation that may be more closely related to coat color than other regions.

Clearly, there is much more experimentation necessary to accomplish the overall goals of the laboratory. While I am thrilled to have obtained promising data for the lack of an association of variation in the attractin gene with coloration in the Kenzin population, I am even more excited to begin the next portion of the experiment as I begin to examine a new population of mice.