The Attractin Gene

A central goal in understanding evolution is determining the genetic basis of adaptation. Thus, this research project will focus on further understanding the role of the agouti gene in the pigmentation of rock pocket mice, Chaetoipus intermedius. The agouti gene has many pleitropic effects, as it influences pigmentation and causes obesity, neurological defects, diabetes, hyperphagia, hyperglycemia, hyperinsulinemia, coronary heart disease, carcinoma, tumorigenesis, and other fatal or debilitating ailments in many mammals, including humans. Although there is great difficulty in understanding evolution due to the lack of information regarding the genetic basis of adaptation, the study of rock pocket mice, Chaetoipus intermedius, has begun to genetically explain adaptation. Rock pocket mice serve as the first demonstration of the genetic basis of adaptive melanism (dark coloration) in mammals. Most rock pocket mice live on light rocks and have light coats; however, when found on dark lava flows, these mice have dark coats. This supports the connection between coat pigmentation and defense against predation, proving this trait essential to the mice’s fitness. While the Mc1r gene causes melanism in one mouse population, other genes, such as agouti, have caused different pigmentations in other populations; thus, this research also exemplifies convergent evolution, as multiple “genetic solutions” have led to the same result within this single species. However, while the genes responsible for pigmentation are known, the mutations within the genes are as of yet undetermined. The only true way to understand evolution is to understand the complete process of adaptation from mutation to phenotype to fitness.

I have been working with Professor Nachman's laboratory since September. In that time, I assisted in the sequencing of the agouti gene. I learned various laboratory methods and took part in all processes leading to the sequencing of a region of the gene. These processes incude designing primers to bind to and amplify a region of the gene through PCR; running and optimizing polymerase chain reaction (PCR), the method of amplifying a region of the gene; running a gel electrophoresis to ensure the PCR product is the desired product by determing the length of the product (this is done by allowing DNA to move from the negative to positive end of an agarose gel, separating the DNA segments by length with short fragments traveling farther than long fragments); cleaning the PCR product and nanodropping the product to determine the concentration of DNA; and finally sending the sample for sequencing. I also learned how to prepare mice specimen and remove the liver and kidneys; I then extracted and cleaned DNA from these organs.

In contrubution to the greater goal of the laboratory in studying natural selection acting on the agouti gene, I am currently conducting my own project of examining the attractin gene. The attractin gene has many variations. One such variation has a sequence similar to the mahogany protein in mice, which is involved in controlling obesity. Another transcript is involved in immune cell clustering that may regulate chemotatic activity (the movement toward or away from a chemical stimulus). Attractin is located on the same chromosome as agouti and may influence the pathways that cause coat color. Thus, in order to understand the evolution of coat color through examination of the agouti gene, one must understand what role, if any, attractin plays in the production of coat color. To this end, I am determining whether variations in the attractin gene are associated with variations in coat color within populations of rock pocket mice that vary in coat color.

To begin the examination of the attractin gene, I mapped the gene, citing the lengths of the introns and exons within the gene. I then identified areas of high conservation in the exons between mice, humans, and kangaroo rats with intervening gene sequences (introns) of less than 2kilobase pairs. I also designed and ordered primers to sequence regions containing portions of exons (expressed gene sequences) 17 and 18 and intron 17 between the two exons. Today, I ran a PCR to amplify this desired region. However, when performing the gel electrophoresis to determine if the product is of desired length, there was some nonspecific DNA. I then ran a gradient PCR, which alters the temperature at which the primer attaches to the separated DNA strands, on a single DNA sample. I analyzed the PCR product using gel electrophoresis and determined the ideal temperature for a PCR reaction involving the chosen primers. Tomorrow, I can run through an entire process for sequencing a region of a gene (as described in the previous paragraph) as many times as possible to complete this process on as many samples as possible.

My day at the laboratory ended with a speaker presentation. Every Monday, an invited speaker is flown to the University of Arizona to speak to the individuals involved in the Ecology and Evolutionary Biology department. This week's speaker was a philosopher discussing the accuracy of natural selection and the reasons for the conflicting opinions on natural selction. His detailed analysis of his topic combined with his British accent and great sense of humor allowed for an enjoyable afternoon.

I have found it an amazing experience, thus far, to take part in actual science and discovery and to be involved in a progressing field. This was my first full day at the laboratory and it proved to be fruitful to spend an uninterrupted day working on data collection. The laboratory community is very close and encouraging. Most importantly, I am ecstatic to soon be obtaining gene sequences!