Abstract
Amylase is an enzyme produced by the salivary glands and pancreas of Homo Sapiens to hydrolyse polysaccharides such as starch and glycogen (Tracey 2019). Variation in the number of AMY1A gene copies is thought to be a result of ancestral heritage and levels of consumed starch (Tracey 2019). Results from a previous study conclude there to be a positive correlation between the number of AMY1A gene repeats and salivary amylase protein expression (Perry et al. 2007). Given these findings, the objective of this study was to determine a possible association between enzyme production, gene copy number, and gene evolution (Tracey 2019). From the study conducted by Perry et al. in “Diet and the evolution of human amylase gene copy number variation”, it was suggested that individuals originating from high-starch diet ancestral populations in arid environments would have a higher number of AMY1A gene copies than individuals from low-starch diet ancestral populations due to increased starch consumption (Perry et al. 2007). The testing of this hypothesis involved various methods used including DNA extraction, gel electrophoresis, amylase assay, and gel imaging. Ultimately, the results were found to be inconsistent with the hypothesis and presented no evidence for an association between enzyme production, gene copy number, and gene evolution.
Introduction
Enzymes are proteins that act as catalysts during biochemical reactions that serve numerous functions in the human body (Tracey 2019). The breakdown of food and starch is an incredibly important enzyme-driven process for human beings. Salivary amylase or alpha amylase (α – amylase) is a glucose-polymer cleavage enzyme responsible for the hydrolysis of α-1,4-glycosidic linkages in polysaccharides (des Gachons and Breslin 2016). It is however unable to hydrolyse α-1,6-glycosidic linkages (Tracey 2019). Amongst digestive enzymes, salivary α-amylase is the most abundant protein found in human saliva (des Gachons and Breslin 2016). In Homo Sapiens, amylase is mainly produced by the salivary glands and pancreas (Tracey 2019). While both salivary and pancreatic amylases are similar in function, both are coded for by different genes with the AMY1 gene found on chromosome 1 (Morris et al. 2016) producing salivary amylase and AMY2 gene producing pancreatic amylase (des Gachons and Breslin 2016).
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Human evolution is characterized by a number of factors including dietary shifts (Perry et al. 2007). With starch having comprised a significant portion of the typical human diet both ancestrally and in present (des Gachons and Breslin 2016), one might infer there to be a relationship between amylase production and human evolutionary history (Perry et al. 2007). The purpose of this investigation was to determine if there was an association between enzyme production, gene copy number, and gene evolution. Prior research has shown there to be a correlation between the number of AMY1 gene repeats and amount of amylase produced suggesting that as AMY1A gene copy number increases, so does the amount of amylase produced (Tracey 2019). This relationship may serve to answer why a large degree of variation exists in the number of AMY1 gene copies amongst individuals (Tracey 2019). With the possibility of selective pressures having acted upon the production of salivary amylase (Perry et al. 2007), it is thought that starch content in diet across different populations in the world is the cause for the variation observed in the AMY1A gene. Results from the study conducted by Perry et al. “Diet and the evolution of human amylase gene copy number variation” suggested that individuals with higher levels of AMY1 gene copies had originated from ancestral agricultural societies or hunter-gatherers in arid environments where they consumed more starch as compared to individuals with low numbers of AMY1 gene copies who originated from rainforest and circum-arctic hunter-gatherers with diets consisting of less starch (Perry et al. 2007).
Based upon these conclusions and the leading hypothesis, the subject (Aqeel Mansuri; 400194043) hypothesized their salivary amylase concentration and AMY1 diploid gene number to be significantly higher than the mean due to their ancestral origin tracing back to South Asia which was presumably arid in environment and consumed high levels of starch. Throughout the investigation, the variable measured was the salivary amylase concentration which was dependent on the number of AMY1A gene copies. The variables were measured in a variety of methods including the creation of a calibration curve to measure the subject’s amylase concentration.
Using the relationship between salivary amylase concentration and number of AMY1A gene copies, as well as the conclusions formed in the study by Perry et al. “Diet and the evolution of human amylase gene copy number variation” as a basis, the rest of the report provides information on the methods used and results obtained in an attempt to validate the leading hypothesis.
Discussion
Variation exists in the number of AMY1A gene copies an individual may have (Tracey 2019). The purpose of this lab was to determine if there was any association or correlation between enzyme production, gene copy number, and gene evolution. Results from the study done by Perry et al. “Diet and the evolution of human amylase gene copy number variation” set the basis of the leading hypothesis as their study suggested that individuals who originated from ancestors that lived in arid environments and consumed high levels of starch would contain a correspondingly increased amount of AMY1A gene copies due to needing a high level of salivary amylase to break down starch (Perry et al. 2007). This also suggested there being a positive correlation between the number of AMY1A gene copies and amount of amylase produced (Tracey 2019). Based upon these results from the study, the subject hypothesized their own salivary amylase concentration and AMY1A gene number to be higher than the mean due to their ancestral ethnicity of South Asia presumably being arid in environment and consuming diets with large amounts of starch.
In the Lab 3 portion of the overall experiment, a scatter plot or standardized calibration curve was created based on the absorbance values of 6 known standards of differing concentrations of amylase. The calibration curve was used to determine the concentration of an unknown substance to known standards (Tracey 2019). This unknown substance was salivary amylase. In the lab 4 portion, a serial dilution consisting of 3 different dilutions (10X, 100X, and 1000X) of the subject’s saliva (Table 1) was created in order to obtain a measurable result of the amylase present in the subject’s saliva (Tracey 2019). Of the 3 absorbance values obtained, the ABS value of 1.12 (corresponding to 10X) was the only value that was present on the trend line from the calibration curve. The salivary amylase concentration of the subject was determined to be 0.0536 mg/ml (Calculation 1).
During gel electrophoresis (Lab 7), a 100bp ladder was loaded onto agarose gel and used to estimate the distance migrated of the subject’s PCR sample. From the semi-log graph the subject’s Actin band was 350bp and migrated 60mm and the amylase band was 160bp and migrated 72mm. The amylase band migrated farther due to its smaller size (in bp). However, the actin and amylase bands of the subject could not be viewed in the gel electrophoresis image, thus the subject was not able to quantify their own DNA fragments. As a result, the values and data shown and Table 3 are of another subject. The reason for the lack of actin and amylase bands is due to errors in the polymerase chain reaction (PCR). It is likely the case that before gel electrophoresis, the subject forgot to add both Primer mix ACTBL2 and Primer mix PER in their reaction mixtures which would have effectively amplified a segment of the subject’s Actin Beta Gene and Salivary Amylase Gene, respectively (Tracey 2019). Without the primers, neither bands were visible in the gel image, the adjusted volume values of both DNA fragments could not be calculated and the subject’s AMY1A gene copy number could not be determined. Nonetheless, was plotted all the subjects AMY1A gene copy numbers against their salivary amylase concentrations. The coefficient value (r) was determined to be -0.0303618 signifying a weak negative correlation between the two variables.
The purpose of this lab was to determine if there was an association between enzyme production, gene copy number, and gene evolution. The results obtained refute the conclusion made by Perry et al. about a positive correlation existing between the number of AMY1 gene copies and amount of amylase produced. The results prove to be inconsistent with the notion that individuals originating from high-starch diet ancestral populations in arid environments would have a higher number of AMY1A gene copies than individuals from low-starch diet ancestral populations due to increased starch consumption. It can be concluded that there is little (weak) to no association between enzyme production, gene copy number, and gene evolution.
A number of errors within the lab may have influenced the difference in final results obtained. First, there were errors with tools of measurement including the spectrophotometers where on several occasions, the ABS values would consistently fluctuate impacting the ABS values obtained. This could be due to the failure correctly calibrate the spectrophotometer. To add, a software “Bio-Rad Image Lab” was used to measure the adjusted volumes of both actin and amylase. This proved to be quite difficult as the highlighting of the bands were to be precise, yet the gel image displayed by the software was quite blurry. This may have led to inaccurate adjusted volume values and thus incorrect values for the number of AMY1 gene copies affecting the final correlation value. Finally, many subjects were unsure of their ancestral lineage which lead to inaccurate estimations of one’s ancestral starch diet. This ultimately may have had an effect on the final correlation calculated.
In terms of future research, it is highly recommended to increase the sample size of the lab in order to obtain more variance amongst results that may be applied to a greater population. It is also suggested to have subjects’ DNA tested for their ethnic or ancestral lineage rather than having them guess. This would greatly improve the accuracy and precision of the overall lab.
Literature Cited
- des Gachons CP, Breslin, PAS. 2016. Salivary amylase: digestion and metabolic syndrome. Curr. Diab. Rep. [accessed 2019 March 27] 16(10):7p 102. https://link.springer.com/article/10.1007/s11892-016-0794-7 doi: 10.1007/s11892-016-0794-7
- Morris J, Hartl D, Knoll A, Lue R, Michael M, Berry A, Biewener A, Farrell B, Holbrook N.M. 2016. Biology: How Life Works Second Edition. New York (NY): W. H. Freeman and Company.
- Perry GH, Dominy NJ, Claw KG, Lee AS, Fiegler H, Redon R, Werner J, Villanea FA, Mountain JL, Misra R, et al. 2007. Diet and the evolution of human amylase gene copy number variation. Nat. Genet. [accessed 2019 March 27]; 39(10):1256-1260. https://www-nature-com.libaccess.lib.mcmaster.ca/articles/ng2123 doi: 10.1038/ng2123
- Tracey A. 2019. Biology 1A03 Laboratory Manual/Notebook. Plymouth (MI): Hayden-McNeil.
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